CN1560232A

CN1560232A - Producing basic proteinase by vibrio metschnikovii DL 33-51 strain

Info

Publication number: CN1560232A
Application number: CNA2004100236701A
Authority: CN
Inventors: 江晓路; 梅承芳
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2004-03-10
Filing date: 2004-03-10
Publication date: 2005-01-05
Anticipated expiration: 2024-03-10
Also published as: CN1281737C

Abstract

The invention relates to a microbe Vibrio metschnikovii Dl 33-51, able to generate basic proteinase with molecular weight of 29.5 kDa. It provides a strain Vibrio metschnikovii Dl 33-51 coming from the nature and a method of producing basic proteinase by this strain. It separates and purifies a strain Vibrio metschnikovii Dl 33-51 from soda lakes, which has a character of generating proteinase. By Bergey's Manual (the ninth edition), this strain has the shape and physiological and biochemical characteristics basically according with those of the Vibrio metschnikovi. This strain has the characters of wide nutrition demands, easily culturing and short culture time. And the generated basic proteinase has high activity. Use this strain as a strain for producing the basic proteinase has the characters of high activity, good pH value and thermal stability, ability to resist surfactant and oxidant. The basic proteinase can be developed into a new enzyme preparation applied in normal-temperature washing agent, implementing industrialized production.

Description

Produce Sumizyme MP with Vibrio metschnikovii DL33-51 bacterial strain

Technical field

The present invention relates to a kind of microorganism Vibrio metschnikovii DL 33-51 bacterial strain that produces Sumizyme MP.

Background technology

By known to inspection information, the literature search, the bacterial classification that produces Sumizyme MP is mainly derived from genus bacillus and some fungies according to the inventor.Bacillus, Streptomyces, Aspergillus, some bacterial strain among the Pseudomonas all can produce Sumizyme MP.According to uncle Jie Shi Bacteria Identification handbook the 9th edition, the form of bacterial strain of the present invention conforms to the Vibrio metschnikovii (Vibrio metschnikovii) of Vibrio with physiological and biochemical property is most of.

Sumizyme MP uses mainly as detergent additive industrial, requires it keeping high enzyme activity and stability in pH and the temperature range widely, also must merge mutually with the washing powder that contains multiple oxygenant and tensio-active agent.Microbe-derived basic protein enzyme reaction optimal pH is generally 9.0-10.0 at present, and various oxygenants and tensio-active agent all can cause alive decline of enzyme in various degree.The Sumizyme MP optimal pH in Vibrio metschnikoviiDL 33-51 source is 12.0 among the present invention, and the existence of oxygenant and tensio-active agent can not cause enzyme forfeiture alive, can impel enzyme activity to rise on the contrary.

Summary of the invention

The purpose of this invention is to provide a kind of method that derives from the bacterial strain Vibrio metschnikovii DL 33-51 of alkali lake and utilize this bacterial strain production Sumizyme MP, and involved enzyme character.

Separation and purification of the present invention goes out the bacterial strain Vibrio metschnikovii DL 33-51 that a strain derives from alkali lake, and it has the characteristic that produces Sumizyme MP, and depositary institution is called for short CCTCC, and preserving number is M204008.Characteristics of the present invention are:

Separate the Vibrio metschnikovii DL 33-51 bacterial strain of producing Sumizyme MP first.This bacterial strain is the knee shape, Gram-negative, and 0.4-0.7 μ m * 1.3-1.5 μ m, end is given birth to single flagellum (Fig. 1).This bacterial classification derives from the alkali lake environment, be the fermented type carbohydrate metabolism, in salt-free and 10%NaCl peptone water, do not grow, 3% and the 7%NaCl peptone water in well-grown, and sensitization test is positive to O-129, determines that it is Vibrio (the feature contrast of this bacterial strain and Vibrio Vibrio metschnikovii Vibrio metschnikovii sees Table 1) according to uncle Jie Shi handbook the 9th edition.This bacterial strain 24 ℃ of cultivation 24h on the casein substratum form single bacterium colony, bacterium colony oyster white, circle, neat in edge.

It is wide that this bacterial classification has the nutritional requirement scope, easily the short characteristics of cultivation and incubation time.Particularly utilize among the present invention nutrition barren semisynthetic medium this bacterial strain that ferments to obtain high yield of enzyme.Cultivate 40h in fermention medium, the vigor of its fermented liquid neutral and alkali proteolytic enzyme promptly reaches 3268U/mL (measuring down for 30 ℃).With the production bacterial strain of bacterial strain of the present invention as Sumizyme MP, have product activity height, good stability, with short production cycle, characteristics that cost is low, can realize suitability for industrialized production.

By separation and purification, SDS-PAGE determines that the molecular weight of the Sumizyme MP that this bacterial strain produces is 29.5kDa (Fig. 2).Its molecular weight is different from the Sumizyme MP of having reported.(table 2).

Property research to this Sumizyme MP shows, the suitableeest enzyme reaction pH12.0, and optimal reactive temperature 55-60 ℃, the following enzyme activity of surveying of this temperature is 11173U/mL.This enzyme has good anti-tensio-active agent and oxygenant characteristic.Insulation is after 1 hour down in 30 ℃ with tween-80 and Trixon-X100, and enzyme work can improve 20% (Fig. 3).H ₂O ₂All work has promoter action (Fig. 4) to enzyme with NaClO.Sumizyme MP with bacterial strain production of the present invention has vigor height under the normal temperature, Heat stability is good, and pH tolerance widely can anti-tensio-active agent and oxygenant, is expected to be applied in the production of normal temperature enzyme-containing detergent as a kind of novel enzyme preparation.

Description of drawings

Fig. 1, the stereoscan photograph of bacterial strain V.metschnikovii DL 33-51.

Fig. 2, the Sumizyme MP SDS-PAGE electrophoresis result of purifying.The left side is pure enzyme list band, and the right is the standard protein molecular weight.

Fig. 3, tensio-active agent is to the influence of basic protein enzyme activity.Under 30 ℃, this enzyme and different concns Triton-X100 and the insulation of tween-80 solution one hour, the per-cent that remaining vigor and control enzyme are lived.

Fig. 4, oxygenant is to the influence of basic protein enzyme activity.Under 30 ℃, this enzyme and different concns NaClO and H ₂O ₂Solution insulation one hour, the per-cent that remaining vigor and control enzyme are lived.

Embodiment

Gather water sample from alkali lake, be chosen at and produce the big bacterium colony of transparent circle on the casein flat board, carry out multiple sieve, obtain having a strain bacterium of high yield alkali protein vigor by measuring its fermentation broth enzyme vigor.This bacterial strain is accredited as Vibrio metschnikovii in the Vibrio by morphologic observation and Physiology and biochemistry.Consisting of of casein substratum: casein 1.0g, bean powder 1.0g, NaCl14.0g, KH ₂PO ₄1.0g, CaCl ₂0.1g, agar 15.0g, H ₂O1000mL, pH10.0.

After bacterial classification activated with the casein substratum, the inoculum size with 5% was linked into 100mL fermention medium (casein 1.0g, bean powder 1.0g, NaCl14.0g, KH is housed ₂PO ₄1.0g, CaCl ₂0.1g, H ₂O1000mL, in 250mL triangular flask pH10.4), 26 ℃ of shake-flask culture 40h, 4 ℃ of following 3000rpm of nutrient solution removed thalline in centrifugal 10 minutes, obtained fermenting enzyme liquid.The condition of enzyme activity determination: 100 μ l enzyme liquid join in the 5mL0.5% casein substrate, are incubated 5min under the certain temperature, add 10% trichoroacetic acid(TCA) termination reaction, filter, and filtrate is colorimetric under 275nm.It is a unit of activity (U) that the enzyme amount that per minute catalysis under the temperature produces 1 μ g tyrosine is measured in definition.

This bacterial classification produces that the zymoprotein that the molecular weight determination of Sumizyme MP adopts acetone precipitation to obtain is dialysed in the damping fluid of 50mmol glycine-NaOH pH10.0, after the drying, through Q-sepharose anion exchange chromatography and the separation and purification of Sephacryl S-100 gel filtration chromatography.SDS-PAGE obtains a single band, and molecular weight is 29.5kDa.

The evaluation contrast of table 1 Vibrio sp.DL 33-51 bacterial strain and V.metschnikovii

Vibrio?sp.DL?33-51 V.metschnikovii

3-12 root flagellum--

Have on the solid medium side hair--

Move about--

Direct rod shape-d

The PHB accumulation-

Pigment--

The two hydrolysis of arginine+-

Oxydase--

Reduction nitrate--

Luminous--

From D glucose aerogenesis--

The V-P generation++

Growth needs Na ion-d

The organic factor-the d of growth needs

Be grown in: 4 ℃--

30℃ + +

35℃ + +

40℃ - +

Amylase++

Gelatinase++

Lipase++

Molten algae enzyme-

Chitinase+

Utilize: acetate++

Aconitate d

The L-arginine-+

Ethanol-

Maltonic acid salt++

The L-glutaminate+

The D-glucuronate-

The DL-glycerinate-

Glycine--

The DL-lactic acid salt+

Lactose-d

The L-leucine-

D-N.F,USP MANNITOL++

D-seminose+d

Melibiose-

The L-proline(Pro)++

Saligenin-

The L-Serine++

D-sorbyl alcohol d

Sucrose++

Trehalose++

L-tyrosine-

The D-wood sugar--

Maltose+

Fluoresce-

The comparison of table 2 Vibrio metschnikovii DL 33-51 strain enzyme-producing and other strain enzyme-producings

The bacterial classification enzyme is molecular weight kDa

Scedosporium?apiospermum 33

Vibrio?parahaemolyticus 43

Bacillus?mojavensis 30

Vibrio?pacini?X4B-7 27

Bacillus?subtilis 44

Shewanella?sp.PA-43 76

Kurthia?spiroforme?sp.nov. 8

Tritirachium?album 33

Pseudomonas?sp.7-11 30

Vibrio?metschnikovii?DL?33-51 29.5

Claims

1, Sumizyme MP is produced bacterial classification Vibrio metschnikovii DL 33-51 and implement patent protection.

2, implement patent protection to utilizing Vibrio metschnikovii DL 33-51 to produce Sumizyme MP.