JPS58155088A - Novel cellulase and its preparation - Google Patents

Abstract

PURPOSE:To prepare a decomposition enzyme reacting specifically with natural cellulose and cellobiose, by culturing a novel cellulase A-producing microbial strain belonging to Ceratocystis genus, and separating the novel cellulase A from the cultured product. CONSTITUTION:A microbial strain belonging to Ceratocystis genus and capable of producing and accumulating cellulase A in high titer in the cultured product, is cultured in a medium containing various saccharides such as sucrose, glucose, starch, etc. as carbon source, and organic materials such as peptone meat extract, etc. as nitrogen source. The obtained cultured liquid is filtered together with a filter aid or centrifuged to obtain crude enzyme liquid. The cellulase A obtained by the purification of the crude product is reactive specifically with natural cellulose and cellobiose, breaks their beta-1, 4-glucoside bonds from their terminals, and has the substrate specificity to break the carboxymethyl cellulose and ''Avicel '' at the middle of the molecule in a short time.

Description

[Detailed description of the invention] Ceratocystes (C@r・tocyst)
ls) culturing a novel cellulase A-producing plate belonging to the genus
This invention relates to a method for producing a new cellulase, which involves separating and collecting the new cellulase from the culture.

In the present invention, "cellulase" refers to an enzyme having the physical and chemical properties described below.

The cellulase obtained according to the present invention has a specific effect on natural cellulase and cellopaiose.
An enzyme with substrate percent isomerism that cleaves the I-darcoside bond from the terminal (exo at) and internally cuts the calcium oxymethyl cellulose (CMC) and aviryl into the inside (endo type) in a very short time of about 60 minutes. It is. Cellulases with a characteristic radical isomerism such as genus have not been found among the conventional cellulases originating from fungi, odontocetes, molluscs, and higher fauna. Therefore, the lulase of the present invention is a novel enzyme in terms of substrate isomerism.

The following is a detailed explanation of the book @ Ming.

First, the organism used in the present invention is Ceratocystes (C@ra-tocystes) which has cellulase A production w@.
stls) It is a l1 species belonging to @, and one example is t.

Ceratocystis densiflora knob sp.
nov. mp. ) (hereinafter referred to as [Ceratocystis AIiJ
It is called. ) is produced.

The Ceratocystis lI4s was isolated from the inside of the wood, which had become soft and viscous, and was published by Institute of Industrial Science and Technology II.
It was deposited with the Institute of Technology on February 26, 1980, and its microbial accession number 636S (rtru
ap-b8! ;).

What are the mycological properties of the Ceratocystes reticulata? It is.

(a) In each medium. ■Growth conditions ■Zupek agar medium (2) Zupeck IP mother extract/pedetone medium produced by Kuwabetsu Shunsakagi sr imitators and ligatured tumors" The medium used for autonomic properties (Zupek agar crucible) Since the growth is insufficient in F.

■Pine l #shochu medium (!) Physiological properties■ Optimum growth conditions plspliA, fiitt: 0℃ ■Growth range pu: pi A, , t~g, 5 temperatures 〇~J
j'C Based on the above properties, the above-mentioned Seratocystis sp.
T, R*Nafl Raj and 5n to 8oycs
drlck :1^ Monograph o4
Chalara and ＾lII＠d （１＠
n@ra'Wllfrid Laurler Un
lv Pr*ss, C5nada (/97!-)), 9 results of sentence comparison search, #4 RakaK
As we found a tree that matched the knowledge of Soki, we concluded that it is appropriate to set it as a new cypress that belongs to Ceratocystis JIIK.

The Ceratocystis genus-Fi produces and accumulates a large amount of cellulase in its culture solution, and in situ, it is used as a carbon source in the culture medium.

Various carbohydrates such as glucose, # powder, -kj%/4-su, I-arm 9〆-1 acupuncture, and peptone as a *g source.

It is preferable to use organic substances such as meat extract, -methane liquor, and fatty soybean, and inorganic substances such as ammonium salt, phosphates, and nitrates can also be used. Others include common inorganic metal salts, vitamins, yeast extract, etc.
It is recommended to add S. Mafc, culture bugs are 20-23
It is preferable to culture at around ℃, but it is also possible to culture with shaking or aeration with agitation. The accumulation of cellulase is at its peak.

The obtained culture solution tube can be treated with a super-assistant, or the crude enzyme can be separated by centripetal separation.

This 11Ii yeast solution may be used as it is, but for example - known methods such as ammonium salt precipitation method, bath solution precipitation method, dialysis method, etc.
It is possible to use a crude fermentation group which is widely used, or it can be further purified and crystallized by a known method and used as a steel refining enzyme.

First, the activity of the cellulase obtained according to the present invention is measured as follows.

[Measurement method and unit of activity]

/16CMC/I (or /16 Avisel or lOm
The present enzyme @114 is added to M*o-biose), acetate buffer w<pus, ri)o, and srj, and heated at 37°C for 1 hour. Reaction completed*, Nelson-Somogyi method (Ne1
ion - SomogyI m@thod) to quantify reducing sugars. In other words, 1 reaction solution Q @ j ml contains 0 Somogyi sample! rmlt- was added, heated at iooo°C for IO minutes to develop one color, and after cooling, Nelson test cake was added at O and SW.

5- Dilute with distilled water. This is measured colorimetrically at a wavelength of 500°.

The unit of fermentation capacity should be ioo unit if reducing sugar equivalent to 1 da glucose is produced in 7 minutes under the above conditions.

The specific activity of the cellulase of the present invention for cellopiose is io, ooo ~ 0,000 units (un)/? It is. Cellulase has an effect on the physical and chemical properties listed below [Physical and chemical properties of oxygen]...Action Cellulase acts specifically on natural cellulase and cellopaiose, The β-/, $-glucoside bond is hydrolyzed.

12+ Substrate-specific cellulase ^ cleaves the β-/4I-glucoside bonds of natural cellulose and cellophores from the terminal ρ (manufactured by Exo).
C) and cut the inside of Abi-7i at the first time (
endoII). That is, the Ceratocystis sp. was cultured using the P paper layer as the only charcoal bulk,
Reinforcement after 7 days? 'If you add the enemy and the active group Sj using the Somogyi method using CUC as a substrate, it will take 60 minutes and 47 groups (1:)
/A/ :l-S phase @ amount is determined ・The activity of U and /Jjμ is suppressed for the light 17 ropaioses, and together they are separated and the action of the end is triggered by a fast storm. It shows the characteristic substrate growth rate.

(3) Optimum - warping stability ρN range pH 3~g
buffer solution, and 9Ng ~//l-1 glycine (Glyc
lne) buffer solution.

The optimal pif for cellulase is -3.7! i;~b,.

(See attached drawing).

In addition, the residual activity of cellulase^ was investigated after incubation at 30°C for 60 minutes.

pill □ was found to have a stable 9M range from 0 to 9.0.

(4) Measurement method of titer/CMC/ml, acetate buffer CpH3/7) o, sy
Add this enzyme solution/1 and react at 37°C for 7 hours, then add Somogyi reagents O and St to 1 reaction solution o and sm.
Heat at 700°C for 10 minutes to develop color1. After cooling* 0
−! Add rat Nelson's reagent and dilute with 5 iz of distilled water. This is determined colorimetrically at a length of 300 mm (Nelson-Somogyi method).

1fsl Action temperature range The action range of cellulase is in the range of mFiJO to 37°C.

(Conditions for inactivation by 61 plJ and m degrees; Inactivation by p-erosion: Regarding cellulase^, conditions for inactivation by p14c were investigated under the same conditions as for the side chambers in the above-mentioned stable range. 9 results, -9,・
It is deactivated at 0 or more and -3,0 or less.

iI1 degree stability: Regarding cellulase^, the temperature was varied at pM 4.00 to examine the conditions for inactivation by 1itt. As a result, the temperature IIL at which heat inactivation begins was 30°C.

() Elliptical split blade method,.

The culture P solution of Ceratocystis sp.
patite) (antacid calcic) was deposited on the column and eluted with the mild II solution, then phosphate buffer C9M
7.0Km node), 77 de') / J (S@phsdsx) ("3@
9-11ad@X”: Swedish Pharmacia a
) Adsorb onto the column, wash thoroughly with the buffer solution described above, and then elute with the buffer solution containing 0.1M sodium chloride. After elution,
Blogel P -1,0 ("Blogel") equilibrated with acetic acid edge*1
Perform filtration using a column (manufactured by Bioland).

Obtain a purified enzyme preparation of cellulase^.

(Tree Molecular weight cellulase ^ is Sepharose (Sspharom)
Based on the l''kfp filtration method using 6B, the molecular weight is approximately 90.
．． Estimated to be 000, Ma-&, 5O8-7/Rilua i
Based on 97 electrophoresis method, approximately 93,000 ~ ioo, o
It was estimated that oo.

Palpitation) Crystal structure Cellulase creates and destroys protein crystals.

The crystal structure is unknown.

Elemental analysis Cellulase^ has a molecular weight of iF due to its behavior due to r-passage.
J? From the behavior of θ, ooo, and 5DS-7/9 amide by pneumophoresis, the molecular weight is approximately qs, ooo to ioo, o.
However, since it is impossible to find out the characteristics of such a polymeric enzyme even if the elemental content is calculated, this −j determination was not performed.

As described above, according to the present invention, cellulase belonging to the genus Ceratocystis can be cultured.
The novel enzyme cellulase 8 can be advantageously shielded.

The method tP* of the present invention will be explained below using Examples.

Examples include Cbu Q, jf Ft* Mg5O4 (7
-3t *'v, +po4/, 06N No.
Ko・0 Hagerisin! ! -, Yeast Extract Co., St.

1 ton jF, cellulose (P paper strips > 10 t water
[1] The above-mentioned Ceratocystes spp. (Kenko Kenbo Saki 1h363 No.) was inoculated into the ridge medium having the above composition, and the temperature was increased to S°C.

SJ! ! A 1-room, 1-room dormitory was held. Culture-1 was reduced in size five times by the ultraviolet V-filtration method, and then diluted with phosphate buffer (
pa7. Hydro equilibrated with Orc#41IIIJ)
Adsorb onto OXyl @916-tits column,
Elute with the above buffer (-gradient: 0.0 to θ, -3).

Next, the column was equilibrated with scale acid slowing solution (-7, θ to 11#) and applied to the KaN column.

After washing thoroughly with the buffer solution*0. Elute with the above buffer containing sM sodium chloride (concentration gradient: 0.0 to 0 mol). After elution, add acetate buffer (ρ & 13.
! A purified enzyme preparation of ioag cellulase^ (specific activity = -0, θ00 unit/t) was obtained.

[Brief explanation of drawings]

The accompanying drawing is a graph showing the performance of cellulase A according to the present invention.

Claims (1)

[Claims] ■ A novel cellulose having the following physical and chemical properties. ■Action: Acts differently on natural cellulose and cellopaiose,
Its β-I, Darglucosi P bond is hydrolyzed. ■Substrate specificity While the β-l and daglucoside bonds of natural cellulose and hiselopiose are cut off from the terminal Wh (echino type), calhokitsum methyl cellulose (CMC) and Avicel are
V for a very short time of about 0 minutes (has substrate tolerance that is endo-type within it. 0 optimum pit and resting pH
It has a stable pli range tpH da, O~9.0. 0 The optimum temperature for action is #- The optimum temperature for action is in the range of 30 to 3C under the conditions of pH &, θ. 0 conditions for inactivation! ;IC-T” lost f&L, 9m 9.0 or more and 3.0
It loses its foil by the Q1+Pr t filtration method, and about 93.000 by the SOS-7 crylamide electrophoresis method. θOO~100.0θO
shows. +21 Ceratocystes (C@ratocyst l
5) Jdi K III's cellulase production'-
A method for producing a new cellulase, which consists of cultivating the cellulase, separating and collecting the new cellulase from the culture. 131 Ceratocystes (C@rHatto@ystls
) A new cellulase belonging to Ceratocystis edendiflora knob sp.
Cooperative order 1ora nov, sp, ) (@
The manufacturing method according to Claim No. J1.