CN101270347A

CN101270347A - High-temperature and strong base resistant clausii bacillus cereus, excretive proteolytic enzyme and preparation

Info

Publication number: CN101270347A
Application number: CNA2008100728742A
Authority: CN
Inventors: 艾尔肯·热合曼; 邓爱华; 王宁; 易霞
Original assignee: Xinjiang University
Current assignee: Xinjiang University
Priority date: 2008-05-08
Filing date: 2008-05-08
Publication date: 2008-09-24

Abstract

The invention discloses high-temperature resistant and alkali resistant bacillus clausii CGMCC No. 1768, a production method thereof, and a method for producing high-temperature alkali protease and other enzyme preparations with the bacterial strain. The bacillus clausii has excellent tolerance, and can grow under extremely adverse environmental conditions as well as in the environment of NaCl of 0 to 20 percent, PH value from 5 to 14, and 4 DEG C to 85 DEG C. The bacillus clausii can survive in wide scope. By adopting the tolerance of the bacterial strain to extremely adverse environment, to research and develop the metabolites of the bacterial strain is very significant. Industrial production requirement can be met; the quality of enzyme preparations can be improved and stabilized; environmental pollution can be reduced; industry can be promoted to be stabilized and developed quickly by developing high temperature resistant and alkali resistant enzymes with excellent thermostability.

Description

High temperature resistant alkaline gram Lloyd's genus bacillus, excretory proteolytic enzyme and preparation

Invention field

The present invention relates to microbial fermentation and product enzyme field thereof.Specifically, the present invention relates to a kind of gram Lloyd's genus bacillus (Bacillus clausii), excretory proteolytic enzyme and preparation method thereof of high temperature resistant salt tolerant alkali.

Background technology

Sumizyme MP has a huge application potential industrial, in industry such as washing composition, leather manufacturing, food-processing, pharmacy and waste treatment, Sumizyme MP replaces traditional chemical reagent and can significantly improve product quality, save cost, eliminate industrial pollutants such as process hides, alleviate pollution, and in industrial application, have very important effect environment.

In order to obtain the Sumizyme MP of industrial value, recently the alkaline-resisting bacterial strain that derives under the extreme environment has been launched deep research, separablely after deliberation obtain having good thermostability, the Sumizyme MP of oxidation-resistance and wide pH value tolerance, thereby can satisfy industrial needs, promote the fast development of industry.Abroad at continuous seed selection novel bacterial, state scholars such as U.S., day, seal have carried out deep research to the halophile that derives under the extreme environment in decades, through separation obtained having good thermostability, the enzyme of oxidation-resistance and wide pH value tolerance.

Uttam in 1999 etc. separate from water sample and obtain a strain gemma tyrothricin, the suitableeest hydrolysis pH of excretory extracellular enzyme is 10.5, the suitableeest hydrolysis temperature is 37 ℃, 50 ℃, 60 ℃ transformation period is respectively 60 hours, 70 hours (Uttam Chand Baner jee, Rajesh Kumar Sani, Wamik Azmi, Raman Soni.Thermostable alkaline protease from Bacillus brevis and itscharacterization as a laundry detergent additive.Process Biochemistry, 1999,35:213-219).

Reports such as calendar year 2001 Johnvesly separate from have a liking for alkali bacterial strain Bacillus sp.JB-99 and obtain a kind of heat-stable Sumizyme MP, the optimum temperuture of enzyme effect is 70 ℃ of (B.Johnvesly, G.R.Naik.Studies on production of thermostable alkaline protease fromthermophilic and alkaphilic Bacillus sp.JB-99 in a chemically definedmedum.Process Biochemistry, 2001,37:139-144).

Kanekar in 2002 etc. have reported that two strains separate product Sumizyme MP strains A .ramosus and the B.alcalophilus that obtains from alkaline lake, they are respectively 11 and 10 at the optimal pH of 65 ℃ of following excretory proteolytic enzyme, the ability of protein hydrolysate very stable (Kanekar PP all in alkaline range, Nilegaonkar SS, Sarnaik SS, Kelkar AS.Optimization of protease activityof alkaliphilic bacteria isolated from alkaline lake in India.Bioresource Technology, 2002,85:87-93).

Joo in 2003 etc. reported a strain from the mud flat at harbour, Jinsen, the Korea S Xihai sea, screen obtain have a liking for alkali gram Lloyd's genus bacillus (Bacillus clausii) I-52, the optimal pH 11 of its exocrine Sumizyme MP, 60 ℃ of optimum temperutures, under the condition of oxygenant and SDS existence, has high stability, handle (the Joo more than 75% and 110% that still can keep original enzyme to live in 72 hours respectively with 5%SDS and 10%H2O2, H.S., Kumar, C.G., Park, G.C., Paik, S.R.and Chang, C.S.Oxidant and SDS-stablealkaline protease from Bacillus clausii I-52:production and someproperties.Journal of Applied Microbiology, 2003,95:267-272).This bacterial strain can only be grown under alkaline condition, and bacterial strain excretory proteolytic enzyme is Sumizyme MP, has bigger activity under alkalescence, and optimal pH is respectively pH11 and 10.5.

Nedra El Hadj-Ali in 2006 etc. have reported a bacillus licheniformis NH1, the optimum temperuture of the outer Sumizyme MP of excretory born of the same parents is 65-70 ℃, has good stability at pH7-11, optimal pH is 10-11, this enzyme is an ion stabilized enzyme of Ca, industrial detergent had outstanding stability and consistency (Nedra ElHadj-Ali, Rym Agrebi, Basma Ghorbel-Frikha, Alya Sellami-Kamoun, SafiaKanoun, Moncef Nasri.Biochemical and molecular characterization of adetergent stable alkaline serine-protease from a newly isolatedBacillus licheniformis NH1.Enzyme and Microbial Technology, 2007,40 (4): 515-523).

One strain of reports such as Huang Hongying separates the Bacillus licheniformis 011 that obtains from the soil sample of Wan Bei saltings, the optimum temperuture of the outer Sumizyme MP of excretory born of the same parents is 60 ℃, optimal pH is 9.0 (Huang Hongying, Fang Haihong, Liu Aimin, Xia Ying, Lv Zhengbing, the research I. microbiology circular 2001,28 (5) of Zhang Linpu one bacillus licheniformis Sumizyme MP: 20-24).

The Bacillus licheniformis B.L JF-1d optimal reactive temperature of reports such as Peng Dong is 55 ℃, optimal pH is 10.5, has higher thermostability, SDS there is stronger tolerance (Peng Dong, Wang Zhongyan, Hu Cheng, the purification of the research II Sumizyme MP of Hu Yongsong bacillus licheniformis alkali protease and property research industrial microorganism 2000,30 (4): 37-40).

The optimum temperuture that Vibrio metschnikovii DL33 bacterial strain produces Sumizyme MP is 60 ℃, optimal pH is up to more than 12, tensio-active agent and oxygenant had stability preferably, various washing powder had good synergetic property (Mei Chengfang, Jiang Xiaolu, Mu Haijin, the seed selection of Wang Peng salt lake high yield proteolytic enzyme bacterium is educated with condition of enzyme production and is studied, Chinese Marine University's journal natural science edition 2005,35 (4): 613-617).

Along with the research to the extreme environment microorganism, the zymetology feature of the proteolytic enzyme that is obtained is more and more superior.Because SDS and oxygenant etc. are the main components in the washing composition, thereby the proteolytic enzyme that not only requires to be added can also need it that SDS and oxygenant etc. is had extreme stability in the higher activity of high-alkali condition maintenance.Over the past two years, the proteolytic enzyme and the production bacterial strain thereof that SDS and oxygenant etc. are had stability carry out screening study, abroad from Joo in 2003 report a strain can secrete SDS and oxygenant had the bacterial strain of stable proteolytic enzyme after, the new anti-SDS of bibliographical information and the proteolytic enzyme of oxygenant are constantly arranged.Domestic also preliminary study report can be secreted the bacterial strain to the stable proteolytic enzyme of SDS and oxygenant, but the performance of oxidation-resistance and anti-SDS is not high, the main zymologic property of homemade alkaline enzyme is difficult to satisfy the needs of washing industrial development, do not have large-scale commercial exploitation potential, the source of large-scale industry alkalescence enzyme mainly depends on external import.Therefore, China needs constantly deeply the research of the exploitation of extreme microorganism itself and Sumizyme MP.And containing the microorganism of peculiar property in the highly basic hypersaline environments such as deep-sea, alkali lake, sand ground, saltings, its excretory Sumizyme MP will have good thermostability and pH stability, utilize the extreme tolerance of microorganism, research and development extreme microorganism excretory proteolytic enzyme, a kind of energy secretion has good enzymatic property and suitable industrial Sumizyme MP bacterial strain has important significance for theories and using value thereby find out.

Summary of the invention

The high temperature resistant anti-highly basic microorganism because wide saltings, Xinjiang is richly stored with.Research Xinjiang extreme microorganism not only helps to enlarge the extreme microorganism resources bank, also helps the development and utilization of Xinjiang particular surroundings microorganism, thereby the enzyme source of high activity and high stability is provided for industrial production.The present invention takes a sample from the plant effluent of Xinjiang Turfan Prefecture, filter out the high temperature resistant anti-alkaline bacterium of a strain, this bacterial strain energy external secretion Sumizyme MP, this enzyme has good thermostability and pH stability, SDS and oxygenant had extreme stability and consistency, and preliminary fermentation condition has been set up in the production of Sumizyme MP, for the Application and Development of the Sumizyme MP of this bacterial strain has been established theoretical basis.

The invention provides the Lloyd's's genus bacillus strain (Bacillus clausii) of a kind of high temperature resistant anti-alkaline gram, its exocrine proteolytic enzyme and production method thereof.Described Sumizyme MP and conventional Sumizyme MP relatively can be brought into play higher protease activity under high temperature highly basic condition.

The present invention is according to the singularity of the geographical environment in Xinjiang, from the typical saline and alkaline water sample in Xinjiang, carry out cultivation, separation and the screening of microbial strains, obtain a collection of high temperature resistant salt tolerant alkali microorganism strains, and therefrom separate high-temperature alkaline protease-producing bacterial strain, be numbered XJU-5, thereby provide a plant height temperature Sumizyme MP to produce bacterium, it has the good characteristic of producing high-temperature alkaline proteolytic enzyme, through microbiology classification and evaluation, belong to gram Lloyd's genus bacillus (Bacillus clausii).

The present invention also provides high-temperature alkaline proteolytic enzyme, obtains through the concrete zymotechnique that the present invention determines by utilizing bacterial strain of the present invention, and the zymin that this bacterial strain provides is not limited only to a kind of product of high-temperature alkaline proteolytic enzyme.

Simultaneously, the present invention also provides a kind of high-temperature alkaline proteinase gene, by this high-temperature alkaline proteinase gene of clonal expression, and stable preservation in the host cell bacillus coli DH 5 alpha.

The present invention specifically provides the strain of a kind of high-temperature alkaline protease production strain, called after XJU-5, and it can produce high-temperature alkaline proteolytic enzyme.Wait with reference to " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey ' s Manual ofSystematic Bacterio-logy ") the 9th edition and " bacterial system identification handbook commonly used " the XJU-5 bacterial strain is carried out morphology mensuration, Physiology and biochemistry detects, the G+C assay, determines that the XJU-5 bacterial strain is the member in gram Lloyd's's genus bacillus strain (Bacillus clausii).The analysis of 16SrRNA homology, Phylogenetic Analysis and cell fatty acid proximate analysis result show that the XJU-5 bacterial strain is for gram Lloyd's genus bacillus (Bacillusclausii), hereinafter to be referred as B.clausii XJU-5.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Microbial Culture Preservation Commission common micro-organisms center (CGMCC).The address: the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100080, preservation date is on July 31st, 2006, preserving number is CGMCC No.1768.38 ℃ of this strain culturing temperature, 42.5 ℃ of the suitableeest culture temperature; Preferred growth is cultivated down for 38 ℃ and can be obtained single bacterium colony in 10-18 hour in extractum carnis media surface (nutrient media components [g/ml] is 0.5% extractum carnis, 1% peptone, 1%NaCl, 1.8% agar powder).Bacterium colony is little, be yellow-white, thickness, flat-shaped, the edge is irregular, opaque.This bacterial strain does not have mobility, facultative aerobic and produce the Gram-positive spore bacteria of bar-shaped spore (being respectively 0.7-0.85 * 2.5-5.0 μ m), and spore expands and in cell central authorities.Transmission electron microscope results shows that strain X JU-5 does not have flagellum and pod membrane.Hydrolyzed starch, tween 80, polysorbate60, polysorbate40 and polysorbas20 can make solation, nitric acid and nitrous acid reduction take place, and catalase and oxydase experiment all present the positive, and produce polygalacturonase and lipase.And VP reacts (pH10.5), indoles produces experiment and all is negative.Soda acid pH tolerance range (pH5-14) is more widely arranged, and optimal pH is 10.But this bacterial strain salt tolerant scope is respectively 0-20% (w/v) NaCl (pH10).Show by above experimental result, XJU-5 is the bacterium of extremely alkaline-resisting, facultative acidproof and salt tolerant, the most outstanding characteristics are that growth scope is extensive, can be respectively grow under the condition of the NaCl of 0-20% concentration, pH5.5-13.6,4-86 ℃, this bacterial strain can be grown under the dual stress conditions of pH13 and 80 ℃ simultaneously.The G+Cmol% of B.clausii XJU-5 is 43.35%, and by the comparison of BLAST homology, the similarity of the 16S rRNA sequence of the 16S rRNA sequence of XJU-5 and B.clausii kind is the highest, and the fatty acid component of XJU-5 mainly is the unsaturated fatty acids of 15:0.

The present invention has good thermal stability by the Sumizyme MP that B.clausii XJU-5 fermentation obtains, and optimum temperuture is 75 ℃, cultivates for 30-80 ℃ to keep more than 90% of original enzyme work in 2 hours respectively.The optimal pH of this enzyme is 10.5, has pH stability widely, cultivates respectively to keep more than 90% of original enzyme work in 100 minutes under the pH10-13 condition.Proteolytic enzyme of the present invention still is a kind of to SDS, H ₂O ₂, enzyme that TritonX-100 is stable, with 1%SDS, 1%H ₂O ₂, 1%TritonX-100 handle can keep respectively after 48 hours that original enzyme lives 262.2%, 117.5%, 102.5%, this character has very important exploitation potential industrial.This product has possessed technical scale throughput, by the optimization to fermention medium and fermentation parameter, selects cheap common nutritive ingredient for use, adopts conventional single batch fermentation mode, and fermenting enzyme work reaches 65U/ml.Referring to accompanying drawing 2.

The present invention also provides the gene of Sumizyme MP, the homology of the thermally-stabilised alkaline protease gene of its sequence and Bacillus sp. is up to 99%, tangible gene difference is 891,1039,1070,1233 of alkaline protease gene sequence of the present invention, is respectively A/G, A/G, A/T, C/T.In gene host cell-bacillus coli DH 5 alpha commonly used with this gene transgene clonal expression, and genetic stability and preservation in its body.Further should be gene constructed to expression vector pET30a (+), known this carrier of those of ordinary skills contains histidine-tagged, foreign protein that can the fast purifying clonal expression, thereby obtain highly purified high-temperature alkaline proteolytic enzyme, produce this Sumizyme MP of purifying for large-scale industrialization and lay the foundation.

The present invention sets up the system process technology of bacterial screening, evaluation, preservation, rejuvenation and seed selection.

One. bacterial screening: from factory's untreated effluent in Turfan, Xinjiang, take a sample, coat and contain 2.0% Wheat Straw, 0.2%NH ₄NO ₃, 0.2%K ₂HPO ₄, 0.02%MgSO ₄7H ₂The nutrient agar (pH10.5) of O, 0.5% yeast extract paste (g/l) obtained single bacterium colony in 18 hours thereby cultivate under 37 ℃, was connected to purifying bacterial classification in the above-mentioned solid medium with the setting-out of inoculating needle picking list bacterium colony.

Two. strain identification

A. Physiology and biochemistry detects

Method according to " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey ' s Manual of SystematicBacterio-logy ") the 9th edition and Sneath et al. (1986) and Nielsen et al. people such as (1995), shape, size, biochemical reactions to bacterial strain detect, high-temperature alkaline proteolytic enzyme microbial strains B.clausii XJU-5 of the present invention, its biological characteristics is as shown in table 1:

Table 1

The mensuration project	The result	The mensuration project	The result	The mensuration project	The result
The mensuration project	The result	The mensuration project	The result	The mensuration project	The result	Shape	Shaft-like	Salt tolerant scope (%)	0-20	Arginine hydrolase	Do not have
Mobility	Do not have	Hydrolyzed casein	Have	The Methionin lytic enzyme	Do not have	Shape	Shaft-like	Salt tolerant scope (%)	0-20	Arginine hydrolase	Do not have
Mobility	Do not have	Hydrolyzed casein	Have	The Methionin lytic enzyme	Do not have	Spore	Have	Gelatin hydrolysate	Have	Urase	Do not have

Gram	Positive	Hydrolyzed starch	Have	The hydrolysis phenylalanine	Do not have
Gram	Positive	Hydrolyzed starch	Have	The hydrolysis phenylalanine	Do not have	Catalase	Have	The hydrolysis polysorbas20	Have	Utilize citric acid	Do not have
Nitrate reductase	Have	The hydrolysis polysorbate40	Have	Indole reaction	Do not have	Catalase	Have	The hydrolysis polysorbas20	Have	Utilize citric acid	Do not have
Nitrate reductase	Have	The hydrolysis polysorbate40	Have	Indole reaction	Do not have	Temperature range (℃)	4-86	The hydrolysis polysorbate60	Have	Hydrolysis tyrosine	Do not have
The pH scope	5-13.6	The hydrolysis tween 80	Have	Grow in N,O-Diacetylmuramidase	Do not have	Temperature range (℃)	4-86	The hydrolysis polysorbate60	Have	Hydrolysis tyrosine	Do not have

The B.G+C assay

Adopt reversed-phased high performace liquid chromatographic to carry out the G+C assay, concrete grammar is with reference to Lin Wanming " bacteria molecule genetic classification identification method ".The G+Cmol% of high-temperature alkaline proteolytic enzyme microbial strains B.clausiiXJU-5 of the present invention is 43.35%.

C.16SrRNA sequential analysis

Utilize the universal primer of bacterial 16 S rRNA: P1:5 ' AGA GTT TGA TCC TGG CTC AG 3 '; P2:5 ' AAG GAG GTG ATC CAG CCG CA 3 '.Pcr amplification obtains the product about 1500bp, reclaims test kit with dna fragmentation and reclaims product.

Finish determined dna sequence by TaKaRa company, sequencing reaction adopts the two deoxynucleotide chain termination method principles of Sanger, at ABI PRISM ^TMCarry out electrophoresis on the full-automatic sequence instrument of 377DNA Sequencer and read preface.

High-temperature alkaline protease-producing bacterium B.clausii XJU-5 of the present invention, its 16S rRNA sequence is as follows:

1 cttccgatac ggctaccttg ttacgacttc accccaatca tttgtcccac cttaggcggc

61 tggctccata aggttgcctc accgacttcg ggtgttacaa actctcgtgg tgtgacgggc

121 ggtgtgtaca agacccggga acgtattcac cgcggcatgc tgatccgcga ttactagcaa

181 ttccggcttc atgcaggcga gttgcagcct gcaatccgaa ctgagaatgg ctttatggga

241 ttggcttcac ctcgcggttt cgctgccctt tgtaccatcc attgtagcac gtgtgtagcc

301 caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg

361 cagtcacctt agagtgccca actcaatgct ggcaactaag atcaagggtt gcgctcgttg

421 cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc acctgtcact

481 ttgcccccga aggggaagcc caatctcttg ggtggtcaaa ggatgtcaag acctggtaag

541 gttcttcgcg ttgcttcgaa ttaaaccaca tgctccactg cttgtgcggg tccccgtcaa

601 ttcctttgag tttcagcctt gcggccgtac tccccaggcg gagtgcttaa tgtgttaact

661 tcggcactac gggcatcgaa acccctaaca cctagcactc atcgtttacg gcgtggacta

721 ccagggtatc taatcctgtt tgctccccac gctttcgcgc ctcagcgtca gttacagacc

781 agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac cgctacacgt

841 ggaattccac tctcctcttc tgcactcaag ctccccagtt tccaatggcc gctcggggtt

901 gagccccgag atttcacatc agacttaaga agccgcctgc gcgcgcttta cgcccaataa

961 ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg

1021 ctttctggtg aggtaccgtc aaggtaccgc cctattcgaa cggtaccgct tcttccctca

1081 caacagagct ttacgacccg aaggccttcc tcactcacgc ggcgttgctc cgtcagactt

1141 tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag

1201 tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg gtaagccgtt

1261 accttaccaa ctagctaatg cgccgcgggc ccatccccta gtgatagccg aagccatctt

1321 tcaccctctc tccaggtgga gaaagggatt atccggtatt agctccggtt tcccggagtt

1381 atcccagtct aaggggcagg ttgcccacgt gttactcacc cgtccgccgc taacgtccgg

1441 gagcaagctc ccttctgtcc gctcgacttg catgtattag gcacgccgcc agcgttcgtc

This gene is declared registration at Genbank, and its number of including is AY959330.Carry out the BLAST compare of analysis, the homology of this sequence and Bacillus clausii is the highest, is 99%, according to adjacent method of attachment constructing system evolutionary tree referring to shown in the accompanying drawing 1.

D. cellular fat acid constituents and content analysis

Extract bacterium lipid acid, use the U.S. Sherlock of MIDI company automatic bacterial identification systems that this thalline fatty acid component is analyzed, concrete grammar is operated according to the instrument explanation.High-temperature alkaline protease-producing bacterium B.clausii XJU-5 of the present invention, its cellular fat acid constituents and content see Table 2.

Table 2

Fatty acid component	Percentage composition (%)	Fatty acid component	Percentage composition (%)
Fatty acid component	Percentage composition (%)	Fatty acid component	Percentage composition (%)	10:0	0.15	15:0	1.84
13:0 ISO	0.39	16:0 ISO	1.43	10:0	0.15	15:0	1.84
13:0 ISO	0.39	16:0 ISO	1.43	13:0 ANTEISO	0.19	16:0	7.71
14:0 ISO	0.85	17:0 ISO	8.33	13:0 ANTEISO	0.19	16:0	7.71
14:0 ISO	0.85	17:0 ISO	8.33	14:0	2.97	17:0 ANTEISO	8.60
13:0 ISO 3OH	1.76	17:0	0.33	14:0	2.97	17:0 ANTEISO	8.60
13:0 ISO 3OH	1.76	17:0	0.33	15:1 ISO I	0.84	18:1 w9c	1.85
15:1 ANTEISO A	0.57	18:1 w7c	1.20	15:1 ISO I	0.84	18:1 w9c	1.85
15:1 ANTEISO A	0.57	18:1 w7c	1.20	15:0 ISO	35.66	18:0	3.76
15:0 ANTEISO	21.41	19:0 ISO	0.06	15:0 ISO	35.66	18:0	3.76
15:0 ANTEISO	21.41	19:0 ISO	0.06	15:1 w6c	0.11

By above Physiology and biochemistry detection, G+C assay, the analysis of 16SrRNA homology, Phylogenetic Analysis and cell fatty acid proximate analysis, every experimental result can show that the XJU-5 bacterial strain is the member in gram Lloyd's's genus bacillus strain (Bacillus clausii), thereby can be accurately XJU-5 strain classification of the present invention be gram Lloyd's genus bacillus (Bacillus clausii), also set up the classification Identifying micro-organisms a kind of reliably, approach accurately.

Three. culture presevation: utilize the cryopreservation method, under the aseptic condition 80% bacterium liquid and 20% glycerine are mixed, bacterial classification is stored in-80 ℃ the Ultralow Temperature Freezer.

Four. actication of culture: go bail for and deposit strain liquid, slowly thawing takes a morsel and is connected on the solid extractum carnis substratum, after cultivating certain hour under the temperature that is fit to strain growth, continuous activation several, the mensuration that can carry out other experiment of bacterial strain.

Five. rejuvenation of spawn: before measuring enzymic activity, determined the flow process that a cover keeps producing enzyme stability of characteristics bacterial strain screening.

At first by (four) step activated spawn, the bacterium that the inoculation activation is good in screening culture medium and cultivating then detects the transparent circle size around the bacterial strain, chooses strain excellent and is inoculated in and cultivates on the slant medium, and this bacterial strain is used as producing bacterial strain.

The present invention also specifically provides a kind of production method of high-temperature alkaline proteolytic enzyme, and it comprises:

(1) gram Lloyd's genus bacillus B.clausii XJU-5CGMCC No.1768 is carried out the strain activation and culture step;

(2) utilize the activated spawn of as above step acquisition to carry out fermentation step;

(3) the purifying extraction step after the fermentation.

Concrete, the present invention adopts conventional single batch fermentation mode on ferment tank technology, and fermenting enzyme work reaches 65u/ml, continuously ferments if can adopt, and fermenting enzyme work will reach 150-200U/ml.During the fermentation, substratum is the basis of zymotechnique.The present invention utilizes shake flask fermentation that fermention medium is screened and optimize, and bacterial activity is detected in experiment.In line with high yield, principle cheaply, the industrialization pilot scale research that on breadboard basis, has carried out the substratum screening and optimized.B.clausii XJU-5 of the present invention there is no particular requirement to the nutrition source of substratum, and the conventional carbon source, the nitrogenous source that are used for microorganism culturing just can satisfy its fermentative production high-temperature alkaline proteolytic enzyme.B.clausii XJU-5 bacterial strain can glucose, sucrose, Semen Maydis powder, starch etc. are made carbon source, and enzyme is also produced in growth, and thalli growth is the fastest when wherein being carbon source with glucose, but enzyme is lived the highest when adding carbon source.Utilize peptone, casein, urea etc. to can be used as nitrogenous source and use, but peptone is the best enzyme nitrogenous source that produces, the inorganic nitrogen-sourced effect that the product enzyme is had reduction.Metal ion has certain effect to fermentation, and Mg, K ion pair are produced enzyme and had promoter action, select the Mg and the K ion of adding 0.01% and 0.05%.Optimized product enzyme substratum is selected in comprehensively the suitableeest carbon source nitrogenous source experiment, can make and produce the yield of enzyme that bacterial strain reaches maximum.The pH of substratum transfers to the required value of fermentation after adopting 10% sodium carbonate solution of sterilizing to sterilize behind the medium sterilization.

In the fermenting process of the present invention, select shake-flask seed to cultivate activate good gram Lloyd's genus bacillus B.clausii XJU-5 bacterial strain picking one to encircle to be inoculated in the 500mL that the 150mL seed culture medium is housed to shake in the bottle, place 46 ℃, cultivate 16h on the 200r/min shaking table, shake-flask seed inserted on the fermentor tank with 2% inoculum size carried out continuous 5 batches fermenting experiment, can obtain stable fermenting enzyme output, thereby 36～43 ℃ of leavening temperatures have been determined, 42.5 ℃ of preferred leavening temperatures, dissolved oxygen control greater than 30% saturated oxygen degree, is controlled the pH value by adding strong aqua and 4mol/L HCl solution all the time.Stirring velocity 250r/min, fermentor tank are kept 0.1MPa, fermentation period 48-72h, and the work of average fermentation enzyme can reach more than the 50U/mL, and the highest fermenting enzyme work can reach 65U/mL.

The nutrient solution that the present invention obtains fermentation is at 12000rpm, centrifugal 30min under 4 ℃, discard precipitation, supernatant is crude enzyme liquid, adopt ammonium sulfate precipitation dialysis, 1.5 times acetone precipitation, carboxymethyl cellulose CM-52 chromatography, Sephadex G-100 chromatographic separation etc., can further separate making enzyme preparation, obtain the liquid enzyme formulation of 300u/ml-350u/ml, quality product meets national relevant industries standard Q/GB1805.3-93.

The present invention also studies the zymologic property of Sumizyme MP, whether has industrial application potentiality and value with the proteolytic enzyme of determining this invention.Those of ordinary skills are known, and the temperature of reaction of high-temperature alkaline proteolytic enzyme is higher, general temperature of reaction 40-60 ℃.The present invention provides the proteolytic enzyme that utilizes high temperature resistant anti-alkaline gram Lloyd's genus bacillus strain B.clausii XJU-5 to obtain simultaneously, it not only has the feature of general Sumizyme MP, or a kind of Sumizyme MP with good thermal stability, optimum temperuture is 75 ℃, cultivate for 30-80 ℃ and can keep more than 90% of original enzyme work in 2 hours respectively, 85 ℃ of cultivations kept 65% of original enzyme work in 2 hours, and the application of temperature of high-temperature alkaline proteolytic enzyme is raise 20 ℃.The optimal pH of this enzyme is 10.5, has pH stability widely, cultivates respectively to keep more than 90% of original enzyme work in 100 minutes under the pH10-13 condition.To SDS, H ₂O ₂, TritonX-100 has extremely strong stability, with 1%SDS, 1%H ₂O ₂, 1%TritonX-100 handle can keep respectively after 48 hours that original enzyme lives 262.2%, 117.5%, 102.5%.Cu ²⁺, Fe ²⁺, Mn ²⁺Can improve enzyme and live Zn ²⁺Enzymic activity there are not great influence, Mg ²⁺, Co ²⁺This enzyme enzyme is lived to be reduced.This product has possessed technical scale throughput, by optimization to fermention medium and fermentation parameter, and enrichment enzyme molecule in fermentation liquid, and utilize the physico-chemical property of enzyme molecule to carry out the product separation.

The present invention specifically provides a kind of technological line () design of primers, pcr amplification high-temperature alkaline proteinase gene of high-temperature alkaline proteinase gene clonal expression simultaneously and has reclaimed

16SrRNA sequence homology according to starting strain gram Lloyd's genus bacillus B.clausii XJU-5 (CGMCC No.1768), sequence alignment is found out similar alkaline protease gene sequence, utilizes the PCR primer of Primer5 programdesign high-temperature alkaline proteinase gene.

The PCR product has an about 1500bp specific band through 1% sepharose detection, referring to accompanying drawing 7, adopts dna fragmentation to reclaim test kit and reclaims this fragment, and specific operation process belongs to known technology.

(2) the T cloning and sequencing of PCR product

The product that above-mentioned recovery is obtained is by the ligase enzyme effect, links to each other with buying in the pMD19-T of TakaRa carrier, is transferred to intestinal bacteria DH _{5 α}Utilize blue hickie screening positive transformant, extract the plasmid of positive transformant, enzyme is cut the dual high-temperature alkaline proteinase gene that obtains for the clone that changes over to of identifying with PCR, enzyme is cut with PCR result meets the expection clip size and is the positive transformant of high-temperature alkaline proteinase gene, referring to accompanying drawing 7.

Finish plasmid dna sequence by TaKaRa company and measure, sequencing reaction adopts the two deoxynucleotide chain termination method principles of Sanger, at ABI PRISM ^TMCarry out electrophoresis on the full-automatic sequence instrument of 377DNA Sequencer and read preface.

The high-temperature alkaline proteinase gene sequence that obtains is as follows:

ATAATGACGGTTATTGACCATAGGAAGTGGACAAACCACCAAAAATGGCAAATACAGACTCCTTTGCAATCTGCTAGATT

GAATAATAAGAGATTTCTGAAATATCTGCTAGATCGAAAAGTATGTTATTTCTGAAATTTCTAAAATAAAAAGGAGGCTG

AATGGTTTATGAGACAAAGTCTAAAAGTTATGGTTTCGTCAACAGTGGCATTGCTTTTCATGGCAAACCCAGCAGCAGCA

AGCGAGGAGAAAAAGGAATATTTGATTGTCGTCGAACCTGAAGAAGTTTCTGCTCAGAGTGTCGAAGAAAGTTATGATGT

GGACGTCATCCATGAATTTGAAGAGATTCCAGTCATTCATGCAGAACTAACTAAAAAAGAATTGAAAAAATTAAAGAAAG

ATCCGAACGTAAAAGCCATCGAAGAGAATGCAGAAGTAACCATCAGTCAAACGGTTCCTTGGGGAATTTCATTCATTAAT

ACGCAGCAAGCGCACAACCGCGGTATTTTTGGTAACGGTGCTCGAGTCGCTGTCCTTGATACAGGAATTGCTTCACACCC

AGACTTACGAATTGCAGGGGGAGCGAGCTTTATTTCAAGCGAGCCTTCCTATCATGACAATAACGGACACGGAACTCACG

TGGCTGGTACAATCGCTGCGTTAAACAATTCAATCGGTGTGCTTGGTGTAGCACCATCGGCTGACTTGTACGCTGTGAAA

GTTCTTGATCGGAATGGAAGTGGTTCGCTTGCTTCTGTAGCTCAAGGAATCGAATGGGCAATTAACAACAACATGCACAT

TATTAATATGAGCCTTGGAAGCACGAGTGGTTCTAGCACGTTAGAGTTAGCTGTCAACCGAGCAAACAATGCTGGTATTC

TCTTAGTAGGAGCAGCAGGTAATACGGGTAGACAAGGAGTTAACTATCCTGCTAGATACTCTGGTGTTATGGCGGCTGCA

GCAGTTGATCAAAATGGTCAACGCGCAAGCTTCTCTACGTATGGCCCAGAAATTGAAATTTCTGCACCTGGTGTCAACAT

AAACAGCACGTACACAGGCAATCGTTACGAATCGCTTTCTGGAACATCTATGGCAACACCACACGTTGCTGGAGTTGCTG

CACTTGTGAAGAGCAGATATCCTAGCTATACGAACAACCAAATTCGCCAGCGTATTAATCAAACAGCAACGTATCTAGGT

TCTCCTAGCCTTTATGGCAATGGATTAGTACACGCTGGACGTGCAACACAATAAATGCACAATGAAAGGCTGAGACAAAA

CTAGCCAAATGTAATAAAAAAGTTCGGATCATCAAAATAAAATGATTCGAGCTTTTTTCTGTTTGCTTCATGGGCTCATC

TGTTGTTTGATTGGCA

This sequence has 1376bp, open reading frame is from 190-1255bp, on NCBI, carry out the BLAST compare of analysis, the homology of the thermally-stabilised alkaline protease gene of its sequence and Bacillus sp. is up to 99%, tangible gene difference is 891,1039,1070,1233 of alkaline protease gene sequence of the present invention, is respectively A/G, A/G, A/T, C/T.The outer alkaline serine protease gene of the born of the same parents of high-temperature alkaline proteinase gene sequence of the present invention and Bacillushalodurans C-125, the serine protease gene AprM of Bacillus sp also has very high similarity, except that the difference that has above-mentioned four sites equally, proteinase gene of the present invention is C at 197, and the outer alkaline serine protease gene of the born of the same parents of Bacillus halodurans C-125, the serine protease gene AprM of Bacillus sp is T, 424 of proteinase gene of the present invention is G, and the outer alkaline serine protease gene of the born of the same parents of Bacillus halodurans C-125 is A.

The proteic sequence of Sumizyme MP of the present invention is as follows:

1 MRQSLKVMVS STVALLFMAN PAAASEEKKE YLIVVEPEEV

41 SAQSVEESYD VDVIHEFEEI PVIHAELTKK ELKKLKKDPN

81 VKAIEENAEV TISQTVPWGI SFINTQQAHN RGIFGNGARV

121 AVLDTGIASH PDLRIAGGAS FISSEPSYHD NNGHGTHVAG

161 TIAALNNSIG VLGVAPSADL YAVKVLDRNG SGSLASVAQG

201 IEWAINNNMH IINMSLGSTS GSSTLELAVN RANNAGILLV

241 GAAGNTGRQG VNYPARYSGV MAAAAVDQNG QRASFSTYGP

281 EIEISAPGVN INSTYTGNRY ESLSGTSMAT PHVAGVAALV

321 KSRYPSYTNN QIRQRINQTA TYLGSPSLYG NGLVHAGRAT

361 Q*

On NCBI, carry out the BLAST compare of analysis, this albumen belongs to proteolytic enzyme S8 family, molecular weight is 38046 dalton, with the outer alkaline serine protease of the born of the same parents of Bacillus halodurans C-125, the serine protease AprM of Bacillus sp., the thermally-stabilised Sumizyme MP of Bacillus sp 98% similarity is arranged all, 95% homology is arranged with the high alkalinity serine protease of Bacillus sp.no.AH101.

The homology comparison result of alkaline protease gene sequence and protein sequence combines with the feature of the thermostability of Sumizyme MP of the present invention, can determine that proteolytic enzyme of the present invention is a kind of heat-staple alkaline serine protease, and called after HAP1 (thermostable alkaline protease).Alkaline protease gene of the present invention and protein sequence are also declared registration on GenBank, the number of including is DQ868543.

(3) carrier of structure high-temperature alkaline proteinase gene

Relevant technologies personnel know, for efficiently expressing exogenous gene need be connected to efficient expression vector with the enzyme gene that obtains.Among the present invention the high-temperature alkaline proteinase gene is connected on pET-30a-c (+) carrier and further expresses.

By implementing the concrete technical indicator of the present invention, realize content of the present invention, can reach following beneficial effect.

A, directed screening restrain Lloyd's genus bacillus (Bacillus clausii to B.clausii XJU-5 high-temperature alkaline proteinase high-yield bacterial strain through being accredited as, B.clausii), this bacterium is the bacterium of extremely alkaline-resisting, facultative acidproof and salt tolerant, the most outstanding characteristics are that growth scope is extensive, can in the NaCl of 0-20% concentration, pH5-13.6, temperature 4-86 ℃ scope, grow respectively, and can under the dual stress conditions of pH13 and 80 ℃, grow, bacterial strain also has the ability of external secretion amylase, lipase and polygalacturonase.

B, pass through the preferred embodiment of the invention, establish B.clausii XJU-5 and produce the stable zymotechnique of bacterial strain, in conventional single batch fermentation mode, fermenting enzyme work reaches 65u/ml, to continuously ferment, fermenting enzyme work will reach 200-300U/ml, and quality product meets national relevant industries standard Q/GB1805.3-93.

C, clone have obtained the gene of Sumizyme MP of the present invention, and genetic stability and preservation in gene host cell-bacillus coli DH 5 alpha commonly used.Further should be gene constructed to expression vector pET30, known this carrier of those of ordinary skills contains histidine-tagged, foreign protein that can the fast purifying clonal expression, thereby obtain highly purified high-temperature alkaline proteolytic enzyme, for this Sumizyme MP of large-scale industrialization Development and Production lays the foundation.

Brief Description Of Drawings:

Figure 1 shows that the chart of systematic evolution tree.

Figure 2 shows that the chart of growth curve and product enzyme curve.In the process of enzymatic production, bacterial strain Bacillus clausii XJU-5CGMCC No.1768 of the present invention begins to produce enzyme after through 24 hours cell growth phase, production of enzyme is exponential growth then, and the output of proteolytic enzyme reached maximum value when just the nutrition in the substratum began to lack in 50-70 hour fermentation time.

Figure 3 shows that high-temperature alkaline proteolytic enzyme optimum temperuture curve.

Figure 4 shows that the temperature stability curve of high-temperature alkaline proteolytic enzyme.

Figure 5 shows that high-temperature alkaline proteolytic enzyme optimal pH curve.

Figure 6 shows that the pH beta stability line chart of high-temperature alkaline proteolytic enzyme.

Figure 7 shows that 1.5% agarose gel electrophoresis detects dna fragmentation figure, wherein 1,3 is Maker DL2000, and 2 is the pcr amplification band of high-temperature alkaline proteinase gene, the 4 pcr amplification bands for PCR checking conversion plasmid, 5 is the enzyme slitting band of plasmid, and 6 is Maker DL15000.

Embodiment

Below, for embodiment the present invention is described, still, the present invention is not limited to following embodiment.In addition, in following explanation, if no special instructions, then % all refers to mass percent.

Embodiment 1: cultivation and the screening of high-temperature alkaline protease-producing bacterium B.ciausii XJU-5

1, sample collecting

Collected specimens from the salt buck in Turfan, Xinjiang.

2, the separation of bacterial strain

Preparation plate culture medium (g/l): 2.0% Wheat Straw, 0.2%NH ₄NO ₃, 0.2%K ₂HPO ₄, 0.02%MgSO ₄7H ₂O, 0.5% yeast extract paste, 1.8% agar powder, pH10.5.With above-mentioned substratum with 1.05kg/cm ², 121 ℃, high pressure steam sterilization 20 minutes.

The centrifugal 10min of water sample 8000rpm with gathering abandons supernatant, and with the physiological saline solution precipitation of sterilization, suspension is by 10 ¹, 10 ³, 10 ⁴, 10 ⁵, 10 ⁷Make 5 dilution gradients, getting each extent of dilution sample liquid 0.2mL evenly coats respectively on above-mentioned substratum (pH10.5) flat board, thereby cultivate down for 38 ℃ and obtained single bacterium colony in 18 hours, be connected to purifying bacterial classification in the above-mentioned solid medium with the setting-out of inoculating needle picking list bacterium colony.

3. produce the screening of Sumizyme MP bacterial strain

Make screening culture medium, put on the screening flat board, cultivated 24-48 hour for 38 ℃ with each single bacterium colony of toothpick picking, what form transparent circle on every side is the protease production strain strain, observe and also to select the big bacterium colony of transparent circle, a bacterial strain called after XJU-5 wherein, preservation behind the purifying.

Screening culture medium: peptone 10g, yeast soak powder 5g, sodium-chlor 5g, agar 18g, casein 5g, dipotassium hydrogen phosphate 1g, magnesium sulfate crystals 0.2g and water 1000ml.

Annotate: pH10.0-11.0, with the pH of the 10% sodium carbonate solution adjusting sterilization back substratum of sterilizing.

Embodiment 2: the fermentation culture of high-temperature alkaline protease-producing bacterium B.clausii XJU-5

Substratum is the basis of zymotechnique technology, has carried out the substratum screening and the optimization of industrialization pilot scale research on the basis of laboratory study, is optimized on the basis of basic fermentation culture.

2. basic fermentation culture: peptone 10g, yeast soak powder 5g, sodium-chlor 5g, dipotassium hydrogen phosphate 1g, magnesium sulfate crystals 0.2g and water 1000ml.

(1) enzymatic production Optimum of culture medium

1. carbon source is to producing the influence of enzyme

Selecting no carbon source, glucose, yeast extract paste, Semen Maydis powder, sucrose, starch to replace yeast in the basic culture solution respectively soaks powder and makes different carbon source nutrient solutions, under identical condition, insert the zymogenic bacteria kind and carry out enzymatic production, 38 ℃ of shaking culture 48 hours, stirring velocity is 150rpm, is index with supernatant enzyme size alive.Collect supernatant after the fermentation ends and carry out enzyme biopsy survey relatively.Enzyme is alive the not highest when wherein having carbon source, secondly is Semen Maydis powder, yeast extract paste, and other do not have great influence to producing enzyme.

2. organic nitrogen source is to producing the influence of enzyme

By as above same method organic nitrogen sources such as wheat bran, Semen Maydis powder, extractum carnis, soyflour, peptone, casein, yeast extract paste, urea are detected, wherein yeast extract paste is that nitrogenous source is that enzyme is alive the highest, secondly is peptone, urea, extractum carnis.

3. inorganic nitrogen-sourced to producing the influence of enzyme

To inorganic nitrogen-sourced detections such as sulfuric acid amine, ammonia chloride, SODIUMNITRATE, the result draws inorganic nitrogen-sourced the reduction greatly and produces enzyme by as above same method.

4. metal ion is to producing the influence of enzyme

Same method adds a certain amount of Na, Mg, Mn, Cu, K plasma respectively in nutrient solution, the fermentation back is measured enzyme and lived, and found that to add a certain amount of Na, K, Mg ion, can promote the raising of thalline production and zymogenic rate.

(2) selection of Optimal compositions of fermentation medium and condition

According to basic fermention medium and fermention medium optimization Test,, selected and produced the enzyme higher substratum that compares in line with high zymogenic rate, principle cheaply.Contain the 150ml nutrient solution in the bottle at 38 ℃, shaking of 500ml, get 5ml and cultivate the activation bacteria suspension in fermentation flask, stirring velocity is 150rpm, fermentation period 48-72h.Detect fermentation broth enzyme output,, prove that bacterium throughput meets production requirement when production of enzyme reaches 50u/ml when above.

3. fermention medium: peptone 5g, yeast soak powder 1g, sodium-chlor 2g, agar 18g, dipotassium hydrogen phosphate 0.1g, magnesium sulfate crystals 0.5g and water 1000ml.

Embodiment 3: the measuring method of high-temperature alkaline protease activity

Issue in 1993-07-29 according to Ministry of Light Industry of the People's Republic of China (PRC), and in " People's Republic of China's industry standard " QB/T 1803,1804-93 of 1994-03-01 enforcement, QB/T 1805,1806-93, the general test method of industrial enzyme preparation, to be decided to be 40 ℃, pH be 10.5 to temperature in test process.

1.1 definition

1g solid enzyme powder (or 1mL liquid enzymes), under certain temperature and pH value condition, it is an enzyme activity unit that the 1min hydrolyzed casein produces 1 μ g tyrosine, represents with u/g (u/mL).

1.2 folin's methods

3.2.1 principle

Proteolytic enzyme is under certain temperature and pH condition. and the hydrolyzed casein substrate produces the nitrogen base acid (as: tyrosine, tryptophane etc.) that contains phenolic group, under alkaline condition, Folin reagent (Folin) is reduced, generate aluminium indigo plant and tungsten blue, use spectrophotometry, calculate its enzyme activity.

3.2.2 reagent and solution

3.2.2.1 the preparation of Folin reagent

In 2000ml ground reflux, add sodium wolframate (Na ₂WO ₄2H ₂O) 100g, sodium aluminate (Na ₂MoO ₄2H ₂O) 25g, water 700mL, 85% phosphoric acid 50mL, concentrated hydrochloric acid 100mL, little fiery boiling reflux 10h takes off reflux cooler, adds Lithium Sulphate (Li in stink cupboard ₂SO ₄) 50g, water 50ml and several dense bromine waters (99%), little again 15min that boils, to remove unnecessary bromine (still have green need add bromine water again after cold, boil and remove excessive bromine), cooling adds water and is settled to 1000mL, and mixing filters.It is golden yellow that the reagent that makes should be, and is stored in the brown bottle.

Use solution: a Folin reagent mixes with two parts of water, shakes up.

3.2.2.2 sodium carbonate solution c (Na ₂CO ₃)=0.4mol/L

Take by weighing anhydrous sodium carbonate (Na ₂CO ₃) 42.4g, with water dissolution and be settled to 1000mL.

3.2.2.3 trichoroacetic acid(TCA) c (CCl ₃COOH)=0.4mol/L

Take by weighing trichoroacetic acid(TCA) 65.4g, with water dissolution and be settled to 1000mL.

3.2.2.4 sodium hydroxide solution c (NaOH)=0.5mol/L

Press GB 601 preparations.

3.2.2.5 hydrochloric acid soln c (HCl)=1mol/L and 0.1mol/L

Press GB 601 preparations.

3.2.2.6 buffered soln

A. phosphoric acid buffer (pH=7.5) is applicable to neutral protease

Take by weighing Sodium phosphate dibasic (Na ₂HPO ₄12H ₂O) 6.02g and SODIUM PHOSPHATE, MONOBASIC (NaH ₂PO ₄2H ₂O) 0.5g is dissolved in water and is settled to 1000mL.

B. lactic acid buffer (pH=3.0) is applicable to aspartic protease

First liquid takes by weighing lactic acid (80%-90%) 10.6g, is dissolved in water and is settled to 1000mL; Second liquid takes by weighing Sodium.alpha.-hydroxypropionate (70%) 16g, is dissolved in water and is settled to 1000mL.Use solution to get first liquid 8mL, add second liquid 1mL, mixing dilutes one times, 0.05mol/L lactic acid buffered soln.

C. borate buffer solution (pH=10.5) is applicable to Sumizyme MP

First liquid takes by weighing Sodium Tetraborate (borax) 19.08g, is dissolved in water and is settled to 1000mL; Second liquid weighing sodium hydroxide 4.0g is dissolved in water and is settled to 1000mL.Use solution to get first liquid 500mL, second liquid 400mL mixing, be diluted with water to 1000mL, above-mentioned various buffered soln must be proofreaied and correct with pH meter.

3.2.2.7 10g/L casein solution (i.e. 1% casein solution)

Take by weighing casein 1.000g, be accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution (if aspartic protease then drip) with dense lactic acid 2-3 moistening after, the about 80mL of buffered soln that adds an amount of various appropriate pH, in boiling water bath, heat while stirring, until dissolving fully, after the cooling, change in the 100mL volumetric flask, be diluted to scale with suitable pH buffered soln.This solution is stored in refrigerator, and validity period is three days.

3.2.2.8 100 μ g/mL L-tyrosine standardized solution

A. take by weighing in advance, be accurate to 0.0002g, be settled to 100mL after dissolving with 1mol/L hydrochloric acid 60mL, be 1mg/mL tyrosine standardized solution prior to 105 ℃ of L-tyrosine 0.1000g that are dried to constant weight.

B. draw 1mg/mL tyrosine standardized solution 10.00mL, be settled to 100mL, promptly obtain 100 μ g/mL tyrosine standardized solution with 0.1mol/L hydrochloric acid.

3.2.3 instrument and equipment

3.2.3.1 40 ℃ ± 0.2 ℃ of water bath with thermostatic control.

3.2.3.2 spectrophotometer should meet the regulation of GB 9721.

3.2.4 analytical procedure

3.2.4.1 the drafting of typical curve

A L-tyrosine standardized solution: by preparing as following table.

The pipe number	The concentration of tyrosine standardized solution (pg/mL)	Get the volume (mL) of the cruel propylhomoserin standardized solution of 100pg/mL	The volume (ml) of water intaking
The pipe number	The concentration of tyrosine standardized solution (pg/mL)		The volume (ml) of water intaking	0 1 2 3 4 5	0 10 20 30 40 50	0 1 2 3 4 5	10 9 8 7 6 5

B. get each 1.00mL of above-mentioned solution (must do parallel test) respectively, respectively add 0.4mol/L sodium carbonate solution 5.00mL, Folin reagent uses solution 1.00mL, place 40 ℃ ± 0.2 ℃ water-bath 20min that develops the color, take out, use spectrophotometer in wavelength 680nm, the 10mm cuvette, with the pipe that does not contain tyrosine is blank, measures its absorbancy respectively.With the absorbance A is ordinate zou, and the concentration of tyrosine is X-coordinate, drawing standard curve (this line should pass through zero point).

According to mapping or using regression equation, calculate the amount (μ g) of the tyrosine when absorbancy is 1, be extinction constant K value, its K value should be in the 95-100 scope.

3.2.4.2 measure

(1) preparation of enzyme liquid to be measured

A. take by weighing enzyme powder 1-2g, be accurate to 0.0002g (or imbitition enzyme 1.00mL), damping fluid dissolving with a small amount of this enzyme, and smash with glass stick and to grind, with in the careful impouring volumetric flask of supernatant liquor, add a small amount of above-mentioned damping fluid in the sediment again and so dissolve, smash and grind 3-4 time then, in last all immigrations volumetric flask, be settled to scale with damping fluid, shake up.With four layers of filtered through gauze, filtrate is diluted to proper concn with damping fluid again according to enzyme activity, for testing with (being diluted to tested test solution light absorption value in the 0.25-0.40 scope).

When b. the enzyme powder is measured, extension rate with reference to as following table (liquid enzymes also can with reference to diluting) as following table.

Enzyme unit alive	General times	Dilution for the first time	Dilution for the second time
Enzyme unit alive	General times	Dilution for the first time	Dilution for the second time	20,000	2000	(2g-200.L 100 times)	5mL-100mL (20 times)
30,000	2500	(2g-500.L 250 times)	5mL-50mL (10 times)	20,000	2000	(2g-200.L 100 times)	5mL-100mL (20 times)
30,000	2500	(2g-500.L 250 times)	5mL-50mL (10 times)	40,000	4000	2g-200mL (100 times)	5mL-200mL (10 times)
50,000	5000	2g-500mL (250 times)	5mL-100mL (20 times)	40,000	4000	2g-200mL (100 times)	5mL-200mL (10 times)
50,000	5000	2g-500mL (250 times)	5mL-100mL (20 times)	8.10 ten thousand	10000	(2g-500.L 250 times)	(5.L-200mL 40 times)

(2) measure

A. earlier casein solution is put into 40 ℃ ± 0.2 ℃ water bath with thermostatic control preheating 5min.

B. press the follow procedure operation:

Test tube A (blank) test tube B (the enzyme sample needs to do three parallel samples)

1 enzyme-added liquid 1.00mL puts into 40 ℃ ± 0.2 1 enzyme-added liquid 1.00mL, puts into 40 ℃ ± 0.2

Preheating 2min in the preheating 2min ℃ water bath with thermostatic control in ℃ water bath with thermostatic control

2 add trichoroacetic acid(TCA) 2.00ml (shaking up), and 40 2 add casein 1.00ml (shaking up), 40 ℃ ±

℃ ± 0.2 ℃ of water bath with thermostatic control in preheating 5min; 0.2 preheating 10min in ℃ water bath with thermostatic control

3 add casein 1.00mL shakes up 3 and adds trichoroacetic acid(TCA) 2.00mL and shake up

4 take out static 10min, filter 4 and take out static 10min, filter

5 get 1.00mL filtrate 5 gets 1.00mL filtrate

6 add sodium carbonate solution 5.0mL 6 adds sodium carbonate solution 5.0mL

7 add Folin reagent uses liquid 1.00mL, and 40 7 add Folin reagent uses liquid 1.00mL, 40

℃ ± 20min develops the color in 20min ℃ ± 0.2 ℃ water bath with thermostatic control of colour developing in 0.2 ℃ of water bath with thermostatic control

8 in the 680nm wavelength, surveys 8 in the 680nm wavelength with the 10mm cuvette, surveys with the 10mm cuvette

It is inhaled, and it is inhaled in luminosity in luminosity

3.2.5 calculate

X＝A×K×4/10×n＝2/5×A×K×n.........................(A3)

In the formula: X is the enzyme activity of sample one by one, u/g (u/mL) t

A is the mean light absorbency of sample parallel test one by one;

K-one extinction constant;

The cumulative volume of 4-one reaction reagent, M1;

10 reaction times 10min one by one are in 1min;

N is extension rate one by one.

Gained is the result represent to integer.

3.2.6 result's tolerance

The parallel test relative error must not surpass 3%.

Embodiment 4: temperature is to the influence of high-temperature alkaline proteolytic enzyme enzyme activity

1. reagent preparation

1.1.pH10.5 borax-sodium hydrate buffer solution

First liquid takes by weighing Sodium Tetraborate (borax) 19.08g, is dissolved in water and is settled to 1000mL; Second liquid weighing sodium hydroxide 4.0g is dissolved in water and is settled to 1000mL.Use solution to get first liquid 500mL, second liquid 400mL mixing, pH meter is proofreaied and correct, and is diluted with water to 1000mL.

1.2 0.5mol/L sodium hydroxide solution

Take by weighing 20.0g NaOH with dissolved in distilled water and be settled to 1000mL, final concentration 0.5mol/L.

1.3 1% casein solution

Take by weighing casein 1.000g, be accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution moistening after, add borax-sodium hydrate buffer solution 80mL of pH10.5, in boiling water bath, heat while stirring, until dissolving fully, after the cooling, change in the 100mL volumetric flask, be settled to 100mL.

2. the mensuration of high-temperature alkaline proteolytic enzyme optimum temperuture and result

2.1 enzyme activity determination

Remove and place 10 ℃ respectively, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 75 ℃, 80 ℃, 90 ℃, react 20min under 95 ℃ the temperature, other condition is all identical, and the enzyme activity determination method is specifically with reference to embodiment 3.

2.2 result

Measure the enzyme that compares each temperature and live, enzyme is alive the highest under 75 ℃ the condition, and the optimum temperuture of enzyme is 75 ℃.Each parallel test relative error is lower than 3%.The result is referring to accompanying drawing 3.

3. the mensuration of the thermostability of high-temperature alkaline proteolytic enzyme and result

3.1 enzyme activity determination

Remove and to place 30 ℃ respectively, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 75 ℃, 80 ℃, be incubated 60min, 120min under 85 ℃ the temperature after, measure residual enzyme and live, the enzyme activity determination method is specifically with reference to embodiment 3.

1.3.2 result

After being incubated 60min, 120min between 30-80 ℃, each residual enzyme work remains on more than 80%, and residual enzyme is lived in also having only slightly and descended behind the insulation 120min.Behind 70 ℃ of insulations 60min, 120min, residual enzyme work is 100%, and the character of enzyme is the most stable under this temperature, and enzyme activity significantly descends during greater than 80 ℃, and the result is referring to accompanying drawing 4, and each parallel test relative error is lower than 3%.

Embodiment 5:pH is to the influence of high-temperature alkaline proteolytic enzyme enzyme activity

1. reagent preparation

1.1 acetic acid-Potassium ethanoate damping fluid (pH5.5)

Take by weighing the 1.55ml glacial acetic acid with dissolved in distilled water and be settled to 1000mL, final concentration 0.1mol is made into A liquid; Take by weighing 19.6g KAc with dissolved in distilled water and be settled to 1000mL, final concentration 0.2mol/L is made into B liquid.A liquid 2.4mL ten B liquid 22.6mL dissolved in distilled water, pH meter are proofreaied and correct to 5.5 and are settled to 100mL.

1.2 potassium primary phosphate-dipotassium hydrogen phosphate damping fluid (pH6.0, pH7.0, pH8.0)

Take by weighing 27.2g KH ₂PO ₄With dissolved in distilled water and be settled to 1000mL and be made into A liquid; Take by weighing 45.6g K ₂HPO ₄With dissolved in distilled water and be settled to 1000mL and be made into B liquid.

PH6.0 potassium primary phosphate-dipotassium hydrogen phosphate damping fluid: A liquid 45mL+B liquid 23mL

PH7.0 potassium primary phosphate-dipotassium hydrogen phosphate damping fluid: A liquid 18mL+B liquid 30mL

PH8.0 potassium primary phosphate-dipotassium hydrogen phosphate damping fluid: A liquid 3mL+B liquid 48mL

All use dissolved in distilled water, pH meter is proofreaied and correct to each pH value and is settled to 100mL.

1.3 borax-sodium hydrate buffer solution (pH9.0,10.0,10.5,11.0)

Take by weighing 19.07g Na ₂B ₄O ₇10H ₂O (borax) is with dissolved in distilled water and be settled to 1000mL, and final concentration 0.05mol/L is made into A liquid; Take by weighing 8gNaOH with dissolved in distilled water and be settled to 1000mL, final concentration 0.2mol/L is made into B liquid.

PH9.0 borax-sodium hydrate buffer solution: A liquid 24mL+B liquid 2mL

PH10.0 borax-sodium hydrate buffer solution: A liquid 25mL ten B liquid 22mL

PH11.0 borax-sodium hydrate buffer solution: A liquid 5mL ten B liquid 25mL

1.4. Sodium phosphate dibasic one NaOH damping fluid (pH12.0, pH13.0, pH14.0)

Take by weighing 67.9g Na ₂HPO ₄, with dissolved in distilled water and be settled to 1000mL, several A liquid that are made into of final concentration 0.05mol; Take by weighing 4.0g NaOH with dissolved in distilled water and be settled to 1000mL, final concentration 0.1mol/L is made into B liquid; Take by weighing 40.0g NaOH with dissolved in distilled water and be settled to 1000mL, final concentration 1.0mol/L is made into C liquid.

PH12.0 Sodium phosphate dibasic one NaOH damping fluid: A liquid 50mL+B liquid 28mL

PH13.0 Sodium phosphate dibasic one NaOH damping fluid: A liquid 10mL+B liquid 50mL

PH14.0 Sodium phosphate dibasic one NaOH damping fluid: A liquid 2mL+C liquid 50mL

1.5. 0.5mol/L sodium hydroxide solution

1.6. 1% casein solution

Take by weighing casein 1.000g, be accurate to 0.001g, with a small amount of 0.5mol/L sodium hydroxide solution moistening after, add the above-mentioned various damping fluid 80mL for preparing, in boiling water bath, heat while stirring, until dissolving fully, after the cooling, change in the 100mL volumetric flask, be settled to 100mL.

2. the mensuration of high-temperature alkaline proteolytic enzyme optimal pH and result

2.1 enzyme activity determination

Casein buffered soln with corresponding pH value is substrate and enzyme reaction, and 40 ℃ are reacted 20min down, and other condition is all identical, measures enzyme then and lives, and the enzyme activity determination method is specifically with reference to embodiment 3.

2.2 result

Measure the relatively enzyme size alive of each pH value, it is the highest to live at the pH10.5 enzyme, and the optimal pH of this enzyme is 10.5, and the result is referring to accompanying drawing 5, and each parallel test relative error is lower than 3%.

2.3 the mensuration and the result of the pH stability of high-temperature alkaline proteolytic enzyme

2.3.1 enzyme activity determination

Enzyme liquid with different pH damping fluids (8.0-13.0) dilution, behind 40 ℃ of water bath heat preservation 100min, is measured residual enzyme and lived, and the enzyme activity determination method is specifically with reference to embodiment 3.

2.3.2 result

This shows that this enzyme stability under the pH13.0 condition is the highest, is 80%, and under other pH condition, residual enzyme is lived and also remained on more than 60%.The result is referring to accompanying drawing 6, and each parallel test relative error is lower than 3%.

The enzyme activity ratio of pH between 5.5-6.0 begins to have improved, and be more stable substantially at the enzyme activity of pH10.0-12.0, and the enzyme of pH13.0 stability alive begins to raise.

Embodiment 6: tensio-active agent and oxygenant are to the influence of high-temperature alkaline proteolytic enzyme enzyme activity

1. the selection of tensio-active agent/oxygenant

In order to detect amalgamation and the stability of high-temperature alkaline proteolytic enzyme of the present invention,, select tensio-active agent Triton-X-100, CTAB, Tween20, SDS and oxygenant H for use according to the requirement of national light industry industry washing composition QB 1806-1993 standard to washing composition ₂O ₂, Sodium peroxoborate detects high-temperature alkaline proteolytic enzyme of the present invention.

2. the preparation of tensio-active agent/oxygenant-enzyme liquid

2.1 borax-sodium hydrate buffer solution of pH10.5

2.2 1%TritonX-100-enzyme liquid

Draw TritonX-100 1mL, be dissolved in enzyme liquid and be settled to 100mL.

2.3 1%CTAB-enzyme liquid

Take by weighing CTAB 1g, be dissolved in enzyme liquid and be settled to 100mL.

2.4 1%Tween20-enzyme liquid

Draw Tween20 1mL, be dissolved in enzyme liquid and be settled to 100mL.

2.5 1%SDS-enzyme liquid

SDS takes by weighing CTAB 1g, is dissolved in enzyme liquid and is settled to 100mL.

2.6 1%H ₂O ₂-enzyme liquid

Draw H ₂O ₂1mL is dissolved in enzyme liquid and is settled to 100mL.

2.7 1% Sodium peroxoborate-enzyme liquid

Take by weighing Sodium peroxoborate 1g, be dissolved in enzyme liquid and be settled to 100mL.

2.8 0.5mol/L sodium hydroxide solution

2.9 1% casein solution

3.3 tensio-active agent/oxidizer treatment enzyme liquid

Tensio-active agent/oxygenant-enzyme the liquid for preparing was placed 38 ℃ of following incubations 48 hours.

3.4 the mensuration that enzyme is lived

Take out the enzyme liquid that tensio-active agent/oxygenant was handled in 48 hours, specifically measure enzyme and live with reference to embodiment 3.

3.4 result

Behind 1%SDS treat enzyme liquid 48h, enzyme work is original 2.62 times, with 1%TritonX-100,1%H ₂O ₂Behind the treat enzyme liquid 48h, the basic and original enzyme of enzyme activity is lived identical, and with also keeping 80% of original enzyme work behind the 1%CTAB treat enzyme liquid, and Tween20, Sodium peroxoborate influence this enzyme activity greatly even make a piece complete deactivation.As seen this high-temperature alkaline proteolytic enzyme and SDS, H ₂O ₂, TritonX-100 has good consistency.The results are shown in Table 4, each parallel test relative error is lower than 3%.

The influence that table 3. tensio-active agent/oxygenant is lived to enzyme

Tensio-active agent/oxygenant (1%)	Relative surplus enzyme (%) alive
Tensio-active agent/oxygenant (1%)	Relative surplus enzyme (%) alive	None	100％
Triton-X-100	102.5％	None	100％
Triton-X-100	102.5％	Tween-20	25％
SDS	262.5％	Tween-20	25％
SDS	262.5％	H ₂O ₂	117.5％
Sodium peroxoborate	0％	H ₂O ₂	117.5％
Sodium peroxoborate	0％	CTAB	80％

4 divalent-metal ions are to the influence of high-temperature alkaline proteolytic enzyme enzyme activity

4.1 the preparation of divalent-metal ion-enzyme liquid

4.1.1 0.025%Fe ²⁺-enzyme liquid: take by weighing FeCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.2 0.025%Co ²⁺-enzyme liquid: take by weighing FeCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.3 0.025%Cu ²⁺-enzyme liquid: take by weighing CuCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.4 0.025%Mn ²⁺-enzyme liquid: take by weighing MnCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.5 0.025%Zn ²⁺-enzyme liquid: take by weighing ZnCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.6 0.025%Mg ²⁺-enzyme liquid: take by weighing MgCl ₂Solid 0.025g uses dissolving of enzyme liquid and constant volume in 100mL.

4.1.7 borax-sodium hydrate buffer solution of pH10.5

4.1.8 0.5mol/L sodium hydroxide solution: take by weighing 20.0g NaOH with dissolved in distilled water and be settled to 1000mL, final concentration 0.5mol/L.

4.1.9 1% casein solution

4.2 the mensuration that enzyme is lived

To add various ionic enzyme liquid and carry out enzyme activity determination, and not add any ionic enzyme liquid and be contrast, and specifically measure enzyme and live with reference to embodiment 3.

4.3 result

Compare with not adding any ionic enzyme activity, in enzyme reaction solution, add Cu ²⁺, Fe ²⁺, Mn ²⁺Can improve enzyme and live, enzyme is had activated effect, Zn ²⁺Enzymic activity there are not great influence, Mg ²⁺, Co ²⁺Enzyme is lived to be reduced.The result is referring to table 4, and each parallel test relative error is lower than 3%.

The influence that table 4. divalent-metal ion is lived to enzyme

Ionic concn (w/v) (0.025%)	Enzymic activity (Uml ^-1)
Ionic concn (w/v) (0.025%)	Enzymic activity (Uml ^-1)	Contrast (not adding any ion)	25.088
Fe ²⁺	66.304	Contrast (not adding any ion)	25.088
Fe ²⁺	66.304	Co ²⁺	5.376
Cu ²⁺	26.880	Co ²⁺	5.376
Cu ²⁺	26.880	Mn ²⁺	112.000
Zn ²⁺	24.192	Mn ²⁺	112.000
Zn ²⁺	24.192	Mg ²⁺	7.168

The pcr amplification and the recovery of embodiment 7 alkaline protease genes

16SrDNA sequence homology according to starting strain gram Lloyd's genus bacillus B.clausii XJU-5 (CGMCC.No.1768), find out high bacterial strain of homology and similar alkaline protease gene sequence according to sequence alignment, utilize the PCR primer of Primer5 programdesign high-temperature alkaline proteinase gene.Primer sequence is

PP1：5′ATA ATG ACG GTT ATT GAC CAT 3′；

PP2：5′GCC AAT CAA ACA ACA GA3′。

Add PCR buffer (plus Mg in the PCR reaction system of every 25ml ²⁺) 2.5 μ l, dNTP 2.5 μ l, each 0.5 μ l of PP1, PP2 primer (concentration is 100pM), Taq archaeal dna polymerase 0.3 μ l, the genomic dna 2.5 μ l of bacterial strain B.clausiiXJU-5 (CGMCC.No.1768) add dual water to the 25 μ l of sterilization.The PCR process is: circulation of 94 ℃ of 5min; 94 ℃ of 1min, 54 ℃ of 45s, 72 ℃ of 1min30s, 30 circulations; 72 ℃ of 7min, a circulation.

Sepharose through 1% detects the characteristic band that an about 1500bp is arranged, and referring to accompanying drawing 7, adopts dna fragmentation to reclaim test kit and reclaims this fragment, and specific operation process is referring to its product description.

The evaluation and the analysis of embodiment 8 B.clausii XJU-5 (CGMCC.No.1768) secretory product

1 actication of culture

Go bail for and deposit strain liquid, slowly thawing takes a morsel and is connected on the solid extractum carnis substratum, after cultivating certain hour under the temperature that is fit to strain growth, and continuous activation several, the mensuration that can carry out other experiment of bacterial strain.

2 identify product enzyme culture medium preparation

Prepare the screening culture medium (compound method such as following table) of various enzymes.

Amylase is identified substratum: peptone 10g, yeast soak powder 5g, sodium-chlor 5g, agar 18g, starch 5g, dipotassium hydrogen phosphate 1g, magnesium sulfate crystals 0.2g and water 1000ml.

Polygalacturonase is identified substratum: peptone 10g, yeast soak powder 5g, sodium-chlor 5g, agar 18g, pectin powder 10g, dipotassium hydrogen phosphate 1g, magnesium sulfate crystals 0.2g and water 1000ml.

The lipase substratum: peptone 10g, yeast soak powder 5g, sodium-chlor 5g, agar 18g, starch 5g, dipotassium hydrogen phosphate 1g, magnesium sulfate crystals 0.2g and water 1000ml.

The detection of 3 inulinase-producing activities and result

Put on the screening flat board with the single bacterium colony of the activation of toothpick picking B.clausii XJU-5 of the present invention CGMCC.No.1768, cultivated 24-48 hour for 38 ℃.After cultivating well, the periphery of bacterial colonies on the amylase substratum splashes into several iodine liquid, have transparent circle to occur, thereby bacterial strain of the present invention has the diastatic ability of external secretion.Periphery of bacterial colonies on the polygalacturonase substratum splashes into several 10%CTAB solution, have transparent circle to occur, thereby bacterial strain of the present invention has the ability of external secretion polygalacturonase.Periphery of bacterial colonies on the lipase substratum has haloing to occur, thereby bacterial strain of the present invention has the ability of external secretion polygalacturonase.

Embodiment 9: the purifying of high-temperature alkaline proteolytic enzyme fermenting enzyme liquid

Protein purification usually uses two class isolation technique, i.e. fractional separation and chromatographic technique, and the rough classification isolation technique has (NH ₄) ₂SO ₄Segmentation salting-out process and isoelectric point precipitation, low pressure chromatographic technique then comprise ion exchange chromatography and molecular sieve column chromatography (gel-filtration) or the like.The purification process of high-temperature alkaline proteolytic enzyme of the present invention is as follows.

1. the preparation of crude enzyme liquid: get the 48h fermented liquid in 4 ℃ through the centrifugal 30min of 12000g, remove thalline and residual substratum, collect supernatant liquor

2. ammonium sulfate precipitation and dialysis: the solid (NH that in crude enzyme liquid, adds porphyrize ₄) ₂SO ₄To 90% saturation ratio, stir 30min, spend the night at refrigerator (4 ℃), then in the centrifugal 30min of 12000g.Precipitation is dissolved in certain volume 0.1%CaAr, handles twice with the DEAE-Mierocrystalline cellulose, and with adsorpting pigment, suction filtration is removed Mierocrystalline cellulose.Enzyme liquid carries out the split pole precipitation then, collects 35%-75% (NH ₄) ₂SO ₄The precipitation of saturation ratio, collecting precipitation. precipitation is dissolved in borax-sodium hydrate buffer solution of pH10.0,36h removes ammonium ion through distill water dialysis, through same damping fluid dialysis, obtains water white transparency enzyme liquid again.

3. carboxymethyl cellulose CM-52 chromatography: the post bed is through 0.4mol/L, and pH8-13 boric acid-NaOH damping fluid balance is with the enzyme liquid upper prop that obtains, launch with same buffer, collect the enzyme activity peak, go up preparative chromatography post again again, still use the same buffer wash-out, collect the enzyme activity peak.

4.Sephadex G-100 chromatography: the SephadexG-100 post is with 0.1% calcium acetate balance, the enzyme liquid that obtains through carboxymethyl cellulose CM-52 chromatography precipitates with cold acetone, 4 ℃ of centrifugal 30min of following 12000r, abandon supernatant after centrifugal, precipitation is dissolved in 0.1% little acetic acid calcium, advances post, after treating that enzyme liquid all enters the post bed, with 0.1% calcium acetate wash-out, substep collector speed is that 2ml/min collects protein peak behind the wash-out, and enzyme liquid is in-4 ℃ of preservations.

5. purification result: the colourless enzyme liquid that gets by carboxymethyl cellulose CM-52 and Sephadex G-100 column chromatography at last. the enzyme of measuring this enzyme liquid is lived. and purity improves 9.87 times behind this enzyme purification, and activity recovery reaches 53.2%, and protein recovery reaches 14.1%.

SEQUENCE LISTING

＜110〉Xinjiang University

＜120〉high temperature resistant anti-alkaline gram Lloyd's genus bacillus, excretory proteolytic enzyme and preparation thereof

＜130〉high temperature resistant anti-alkaline gram Lloyd's genus bacillus, excretory proteolytic enzyme and preparation thereof

<160>3

<170>PatentIn version 3.3

<210>1

<211>1500

<212>DNA

<213>Bacillus clausii

<400>1

cttccgatac ggctaccttg ttacgacttc accccaatca tttgtcccac cttaggcggc 60

tggctccata aggttgcctc accgacttcg ggtgttacaa actctcgtgg tgtgacgggc 120

ggtgtgtaca agacccggga acgtattcac cgcggcatgc tgatccgcga ttactagcaa 180

ttccggcttc atgcaggcga gttgcagcct gcaatccgaa ctgagaatgg ctttatggga 240

ttggcttcac ctcgcggttt cgctgccctt tgtaccatcc attgtagcac gtgtgtagcc 300

caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt ttgtcaccgg 360

cagtcacctt agagtgccca actcaatgct ggcaactaag atcaagggtt gcgctcgttg 420

cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc acctgtcact 480

ttgcccccga aggggaagcc caatctcttg ggtggtcaaa ggatgtcaag acctggtaag 540

gttcttcgcg ttgcttcgaa ttaaaccaca tgctccactg cttgtgcggg tccccgtcaa 600

ttcctttgag tttcagcctt gcggccgtac tccccaggcg gagtgcttaa tgtgttaact 660

tcggcactac gggcatcgaa acccctaaca cctagcactc atcgtttacg gcgtggacta 720

ccagggtatc taatcctgtt tgctccccac gctttcgcgc ctcagcgtca gttacagacc 780

agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac cgctacacgt 840

ggaattccac tctcctcttc tgcactcaag ctccccagtt tccaatggcc gctcggggtt 900

gagccccgag atttcacatc agacttaaga agccgcctgc gcgcgcttta cgcccaataa 960

ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag ttagccgtgg 1020

ctttctggtg aggtaccgtc aaggtaccgc cctattcgaa cggtaccgct tcttccctca 1080

caacagagct ttacgacccg aaggccttcc tcactcacgc ggcgttgctc cgtcagactt 1140

tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc cgtgtctcag 1200

tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgttgccttg gtaagccgtt 1260

accttaccaa ctagctaatg cgccgcgggc ccatccccta gtgatagccg aagccatctt 1320

tcaccctctc tccaggtgga gaaagggatt atccggtatt agctccggtt tcccggagtt 1380

atcccagtct aaggggcagg ttgcccacgt gttactcacc cgtccgccgc taacgtccgg 1440

gagcaagctc ccttctgtcc gctcgacttg catgtattag gcacgccgcc agcgttcgtc 1500

<210>2

<211>1376

<212>DNA

<213>Bacillus clausii

<400>2

ataatgacgg ttattgacca taggaagtgg acaaaccacc aaaaatggca aatacagact 60

cctttgcaat ctgctagatt gaataataag agatttctga aatatctgct agatcgaaaa 120

gtatgttatt tctgaaattt ctaaaataaa aaggaggctg aatggtttat gagacaaagt 180

ctaaaagtta tggtttcgtc aacagtggca ttgcttttca tggcaaaccc agcagcagca 240

agcgaggaga aaaaggaata tttgattgtc gtcgaacctg aagaagtttc tgctcagagt 300

gtcgaagaaa gttatgatgt ggacgtcatc catgaatttg aagagattcc agtcattcat 360

gcagaactaa ctaaaaaaga attgaaaaaa ttaaagaaag atccgaacgt aaaagccatc 420

gaagagaatg cagaagtaac catcagtcaa acggttcctt ggggaatttc attcattaat 480

acgcagcaag cgcacaaccg cggtattttt ggtaacggtg ctcgagtcgc tgtccttgat 540

acaggaattg cttcacaccc agacttacga attgcagggg gagcgagctt tatttcaagc 600

gagccttcct atcatgacaa taacggacac ggaactcacg tggctggtac aatcgctgcg 660

ttaaacaatt caatcggtgt gcttggtgta gcaccatcgg ctgacttgta cgctgtgaaa 720

gttcttgatc ggaatggaag tggttcgctt gcttctgtag ctcaaggaat cgaatgggca 780

attaacaaca acatgcacat tattaatatg agccttggaa gcacgagtgg ttctagcacg 840

ttagagttag ctgtcaaccg agcaaacaat gctggtattc tcttagtagg agcagcaggt 900

aatacgggta gacaaggagt taactatcct gctagatact ctggtgttat ggcggctgca 960

gcagttgatc aaaatggtca acgcgcaagc ttctctacgt atggcccaga aattgaaatt 1020

tctgcacctg gtgtcaacat aaacagcacg tacacaggca atcgttacga atcgctttct 1080

ggaacatcta tggcaacacc acacgttgct ggagttgctg cacttgtgaa gagcagatat 1140

cctagctata cgaacaacca aattcgccag cgtattaatc aaacagcaac gtatctaggt 1200

tctcctagcc tttatggcaa tggattagta cacgctggac gtgcaacaca ataaatgcac 1260

aatgaaaggc tgagacaaaa ctagccaaat gtaataaaaa agttcggatc atcaaaataa 1320

aatgattcga gcttttttct gtttgcttca tgggctcatc tgttgtttga ttggca 1376

<210>3

<211>360

<212>PRT

<213>Bacillus clausii

<400>3

Met Arg Gln Ser Leu Lys Val Met Val Ser Ser Thr Val Ala Leu Leu

1 5 10 15

Phe Met Ala Asn Pro Ala Ala Ala Ser Glu Glu Lys Lys Glu Tyr Leu

20 25 30

Ile Val Val Glu Pro Glu Glu Val Ser Ala Gln Ser Val Glu Glu Ser

35 40 45

Tyr Asp Val Asp Val Ile His Glu Phe Glu Glu Ile Pro Val Ile His

50 55 60

Ala Glu Leu Thr Lys Lys Glu Leu Lys Lys Leu Lys Lys Asp Pro Asn

65 70 75 80

Val Lys Ala Ile Glu Glu Asn Ala Glu Val Thr Ile Ser Gln Thr Val

85 90 95

Pro Trp Gly Ile Ser Phe Ile Asn Thr Gln Gln Ala His Asn Arg Gly

100 105 110

Ile Phe Gly Asn Gly Ala Arg Val Ala Val Leu Asp Thr Gly Ile Ala

115 120 125

Ser His Pro Asp Leu Arg Ile Ala Gly Gly Ala Ser Phe Ile Ser Ser

130 135 140

Glu Pro Ser Tyr His Asp Asn Asn Gly His Gly Thr His Val Ala Gly

145 150 155 160

Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pro

165 170 175

Ser Ala Asp Leu Tyr Ala Val Lys Val Leu Asp Arg Asn Gly Ser Gly

180 185 190

Ser Leu Ala Ser Val Ala Gln Gly Ile Glu Trp Ala Ile Asn Asn Asn

195 200 205

Met His Ile Ile Asn Met Ser Leu Gly Ser Thr Ser Gly Ser Ser Thr

210 215 220

Leu Glu Leu Ala Val Asn Arg Ala Asn Asn Ala Gly Ile Leu Leu Val

225 230 235 240

Gly Ala Ala Gly Asn Thr Gly Arg Gln Gly Val Asn Tyr Pro Ala Arg

245 250 255

Tyr Ser Gly Val Met Ala Ala Ala Ala Val Asp Gln Asn Gly Gln Arg

260 265 270

Ala Ser Phe Ser Thr Tyr Gly Pro Glu Ile Glu Ile Ser Ala Pro Gly

275 280 285

Val Asn Ile Asn Ser Thr Tyr Thr Gly Asn Arg Tyr Glu Ser Leu Ser

290 295 300

Gly Thr Ser Met Ala Thr Pro Hi s Val Ala Gly Val Ala Ala Leu Val

305 310 315 320

Lys Ser Arg Tyr Pro Ser Tyr Thr Asn Asn Gln Ile Arg Gln Arg Ile

325 330 335

Asn Gln Thr Ala Thr Tyr Leu Gly Ser Pro Ser Leu Tyr Gly Asn Gly

340 345 350

Leu Val His Ala Gly Arg Ala Thr

355 360

Claims

1, a kind of have high temperature resistant anti-alkaline gram Lloyd's genus bacillus (Bacillus clausii) CGMCCNo.1768.

2, the production method of a kind of high temperature resistant anti-alkaline gram Lloyd's genus bacillus (Bacillus clausii) CGMCC No.1768 as claimed in claim 1 is characterized in that, it comprises,

A: CGMCC No.1768 carries out the strain activation and culture step to gram Lloyd's genus bacillus (Bacillus clausii);

B: the activated spawn of utilizing steps A to obtain is carried out fermentation step;

C: the purifying extraction step after the fermentation.

3, as the production method of high temperature resistant anti-alkaline gram Lloyd's genus bacillus (Bacillus clausii) CGMCCNo.1768 as described in the claim 2, it is characterized in that, in strain activation and culture, this strain growth temperature range is 4-86 ℃, 38 ℃ of culture temperature can be grown under the condition of the NaCl of 0%-20% concentration, pH5.5-13.6 respectively.

4, as the production method of high temperature resistant anti-alkaline gram Lloyd's genus bacillus (Bacillus clausii) CGMCCNo.1768 as described in the claim 2, it is characterized in that, in the fermentation culture step, 36～43 ℃ of leavening temperatures, the saturated oxygen degree is greater than 30%, add strong aqua and 4mol/L HCl solution control pH value, stirring velocity 250r/min, fermentor tank are kept 0.1MPa, fermentation period 48-72h.

5, the production method of high temperature resistant anti-alkaline gram Lloyd's genus bacillus (Bacillus clausii) CGMCC No.1768 as claimed in claim 3, it is characterized in that in the fermentation, 42.5 ℃ of the suitableeest training leavening temperatures of bacterial strain, and can under the dual stress conditions of pH13 and 80 ℃, grow.

6, a kind of high-temperature alkaline proteolytic enzyme is characterized in that, it utilizes the described gram of claim 1 Lloyd's genus bacillus (Bacillus clausii) CGMCC No.1768, is prepared from by the described production method of claim 2.

7, high-temperature alkaline proteolytic enzyme as claimed in claim 6 is characterized in that, this high-temperature alkaline proteolytic enzyme is a kind of heat-staple alkaline serine protease, and its optimum temperuture is 75 ℃, and optimal pH is 10.5.

8, high-temperature alkaline proteolytic enzyme as claimed in claim 7, it is characterized in that, this alkaline serine protease is cultivated for 85 ℃ and was kept 65% of original enzyme work in 2 hours, stability is 80% under the pH13.0 condition, under other pH condition, residual enzyme is lived and is also remained on more than 60%, and the application of temperature of high-temperature alkaline proteolytic enzyme is raise 20 ℃, and to SDS, H ₂O ₂, TritonX-100 has extremely strong stability, with 1%SDS, 1%H ₂O ₂, 1%TritonX-100 handle can keep respectively after 48 hours that original enzyme lives 262.2%, 117.5%, 102.5%, Cu ²⁺, Fe ²⁺, Mn ²⁺Can improve enzyme and live Mg ²⁺, Co ²⁺Can reduce enzyme lives.

9, a kind of sequence as high-temperature alkaline proteolytic enzyme as described in the claim 6 is characterized in that this sequence has 1376bp, open reading frame is from 190-1255bp, 891,1039,1070,1233 of sequences are respectively A/G, A/G, A/T, C/T, and 197 is C, and 424 is G.

10, the aminoacid sequence of high-temperature alkaline proteolytic enzyme as claimed in claim 9 is characterized in that, the molecular weight of this high-temperature alkaline proteolytic enzyme is 38046 dalton, is a kind of heat-staple Sumizyme MP (thermostable alkaline protease).