CN112280768B - Alkaline protease low-temperature mutant and application thereof in sludge treatment - Google Patents
Alkaline protease low-temperature mutant and application thereof in sludge treatment Download PDFInfo
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- CN112280768B CN112280768B CN202011158007.8A CN202011158007A CN112280768B CN 112280768 B CN112280768 B CN 112280768B CN 202011158007 A CN202011158007 A CN 202011158007A CN 112280768 B CN112280768 B CN 112280768B
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C12Y304/21106—Hepsin (3.4.21.106)
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Abstract
The invention provides mutants SPL-12 and SPL-17 of alkaline protease low-temperature PB92, wherein the amino acid sequences of the mutants are SEQ ID NO. 1-3; compared with parent enzymes, the SPL-12 and the SPL-17 provide improved stability and low-temperature performance, and are suitable for the acid production treatment process of alkaline sludge.
Description
Technical Field
The application belongs to the field of protein and the field of environmental protection, and particularly provides low-temperature mutants SPL-12 and SPL-17 of alkaline protease PB92, wherein the amino acid sequences of the low-temperature mutants SPL-12 and SPL-17 are SEQ ID NO. 1-3; compared with parent enzymes, the SPL-12 and the SPL-17 provide improved stability and low-temperature performance, and are suitable for the acid production treatment process of alkaline sludge.
Background
The common methods for sewage treatment all comprise the step of transferring pollutants, organic matters, microorganisms and the like in water into sludge to clean water, so that the treatment of the sludge rich in the pollutants, the organic matters and the microorganisms becomes the most critical and fundamental step in the sewage treatment, the treatment cost of the sludge accounts for more than half of the sewage treatment cost, and the treatment cost is also the root of reaching the standard of pollutant discharge.
At present, the commonly used sludge treatment modes are landfill, natural drying, high-temperature incineration and the like, the methods not only have higher secondary pollution risk, but also increasingly bear the treatment enterprises and governments in cost of land, fuel and the like. In order to reduce the components of sludge treatment and improve the treatment sustainability, the recycling of sludge is a necessary choice, and the anaerobic fermentation treatment of sludge accounts for about 50 percent of the total amount of sludge treatment in developed countries in Europe.
Whether anaerobic fermentation using methane as a product or anaerobic fermentation using organic acids as a product requires hydrolysis to break down microbial cells and extracellular polymers to increase the rate and efficiency of anaerobic fermentation, currently available enzymes include cellulases, hemicellulases, proteases, amylases, lysozyme, etc. (Dai XH et al. Simultaneous fermentation of microbial and methane content in biological free activated sludge and bacterial inorganic co-differentiation: the effects of pH and C/N ratio, Bioresource Technology, 2016, 216: 323-. The protease is the main enzyme for destroying the conglomerates in the sludge and has the function of destroying cells. At present, most of protease used in sludge treatment is neutral and acidic protease, and the effect of alkaline sludge, such as sludge of printing and dyeing enterprises and certain non-ferrous metal smelting processing enterprises, and sludge subjected to alkaline treatment is poor.
Disclosure of Invention
In order to solve the above problems, the present applicant has tried to treat sludge with the conventional PB92 alkaline protease, and found that the protein elution effect and the acid production effect are not preferable, presumably because the enzyme activity is decreased after the action time exceeds 2 hours due to the poor stability of PB 92. Then, the applicant screens the stability variants, particularly the low-temperature stability improvement variants, of PB92 by an error-prone PCR method, and the obtained SPL-12 and SPL-17 are obviously improved in the aspect, and the protein dissolution effect and the acid yield are obviously improved when the variants are used for dissolving sludge.
In one aspect, the present application provides a variant alkaline serine protease which incorporates a mutation selected from the group consisting of S76D, P129E, S130K, G260H, S130A, a166D on the basis of the alkaline serine protease having the amino acid sequence of SEQ ID No. 1.
Further, the stability of the variants is improved.
Further, the mutations are in the group consisting of S76D, P129E, S130K and G260H, or in the group consisting of P129E, S130A and a 166D.
Further, the amino acid sequence of the variant is SEQ ID NO. 2.
Further, the amino acid sequence of the variant is SEQ ID NO. 3.
In another aspect, the present application provides the use of the above variants in sludge treatment.
Further, the sludge is alkaline sludge.
Further, the sludge treatment is fermentation acidogenesis.
Further, the variant treatment of sludge promotes the digestion of proteins and polysaccharides.
The variants in the present application can be produced by molecular biology and fermentation engineering methods well known in the art, and the host bacteria used can be of the species commonly used in molecular biology and fermentation engineering, including but not limited to escherichia coli, bacillus subtilis, yeast.
The sludge in this application can be industrial sludge containing protein and polysaccharide, domestic sludge, agricultural sludge, etc., and particularly preferred are sludge in alkaline form, including but not limited to dye house sludge, nonferrous metal smelting house sludge, and alkaline treated domestic sludge.
Fermentation acidogenic products in the present application include, but are not limited to, acetic acid, propionic acid, butyric acid, valeric acid and derivatives thereof.
The strain for fermentation and acid production in the application can be inoculated from sludge with good fermentation effect, and can also be prepared from pure culture strains according to the research on fermentation strains in the field.
Drawings
FIG. 1 is a graph of enzyme activity-pH curves of parent PB32, SPL-12, SPL-17;
FIG. 2 is the enzyme activity-temperature curve diagram of parent PB32, SPL-12, SPL-17.
Detailed Description
Primary reagents and instruments
The forsin reagent is produced by Shanghai Lianmai bioengineering Co., Ltd;
the spectrophotometer 754N is produced by shanghai instrument electrical analyzer limited;
the error-prone PCR kit GeneMorph II random mutagenetics kit is produced by Agilent;
the BCA protein concentration detection kit is produced by Solarbio;
the concentrated sulfuric acid-phenol sugar concentration detection kit is produced by Shenzhenzekiake biotechnology Limited;
the genetic engineering bacteria and the vector are self-preserved in a laboratory where the applicant is located;
lysozyme was purchased from Beijing Xiaheng Biotech Co., Ltd (label 40000U/mg, optimum pH 6.5);
cellulase was purchased from Xin chemical products, Inc., Ming, Henan (labeling 100U/mg, optimal pH 6.0).
The sludge used in example 3 was collected from a secondary sedimentation tank of a sewage treatment plant in a certain town of Dongguan industrial park (except a small amount of office/domestic sewage in an office building, the main sewage source of the sewage treatment plant is near three printing and dyeing enterprises), and the basic indexes are as follows: TSS13.2-14.8g/L, VSS 10.9.9-12.2 g/L, SCOD 0.16-0.18g/L (digestion method), pH 8.4-8.9;
other reagents and instruments are all of conventional domestic varieties.
Protease activity determination method:
determined using the forskolin method: adding 2mL of 100ug/mL enzyme solution prepared by boric acid buffer solution with required pH into the serial tube; keeping the temperature of the tube at the required temperature for 5 min; adding 1mL of enzyme solution, mixing, and standing for reaction for 10 min; adding 3mL0.4M trichloroacetic acid to terminate; taking 1mL of supernatant, adding 1mL of formalin reagent and 0.4M Na2CO35mL, mixing and placing at 40 ℃ for color development for 20 min; determination of OD660 nm; a standard curve was constructed and enzyme activity was determined based thereon.
The sludge dissolution experiment method comprises the following steps:
adding the sludge sample and the enzyme into a 500mL flask, filling nitrogen, and sealing; shaking at 100rpm at 30 ℃, and sampling and analyzing on time; after centrifugation at 10000g for 10 minutes, the obtained supernatant is used for detecting parameters such as COD, dissolved protein, dissolved polysaccharide and the like.
Soluble protein assay
The detection is carried out by using a bicinchoninic acid method, and the specific process is carried out according to the instruction of the kit.
Dissolved polysaccharide assay
The detection is carried out by using a concentrated sulfuric acid-phenol method, and the specific process is carried out according to the instruction of the kit.
Example 1 mutant acquisition procedure
Error-prone PCR and mutant enzyme library construction
The parent protein used in the present application is alkaline serine protease PB92(SEQ ID NO.1) isolated from Bacillus alcalophilus in the early 90 s of the last century, which is most remarkably characterized by high activity at high pH, product related patents filed by Novitin and related companies, and which have been out of date, PB92 sequence, basic properties and research methods are available from NCBI, related patents and non-patent literature.
The PB92 Gene sequence was synthesized by reference to the prior art (e.g., Cloning, Characterization, AND Multiple Chromosomal Integration of a Bacillus Alkaline Protease Gene, APPLID AND ENVIRONMENTAL MICROBIOLOGY, 1991, vol.57, No. 4); transforming into Escherichia coli; after culturing, taking single colony plasmid for sequencing verification; the plasmid containing the correct sequence was used as template for multiple rounds of error-prone PCR (Agilent GeneMorph II random mutagenesis kit).
The error-prone PCR product was ligated to the vector pET32 a; transformation into BL21(DE3) E.coli; screening positive clones to form a mutant enzyme library; positive clones in the constructed library are cultured on a culture plate for 96 hours in an induction way, the protease activity at pH8.0 and 50 ℃ and the protease activity at pH8.0 and 30 ℃ are measured by taking supernatant, and the protease activity at pH8.0 and 30 ℃ is measured after the supernatant is placed at 30 ℃ for 3 hours.
Screening, expression and sequencing of mutants
The basic activity, the low-temperature activity and the activity maintaining time factor are mainly considered, 22 mutants with the enzyme activity performance meeting the requirement are obtained in three total rounds of screening, and two of the mutants are selected for further practical research after further experiments. Inoculating the genetically engineered bacteria into a shake flask filled with 100ml of LB culture medium, and adding 100uM IPTG (isopropyl-beta-thiogalactoside) for induction culture for 72 hours; centrifuging to obtain crude enzyme solution, dialyzing, concentrating, and purifying with GST to obtain purified parent PB32, SPL-12, and SPL-17; the electrophoresis shows that the molecular weight of the gel is about 28 kDa. Determining the sequence of the polypeptide as SEQ ID NO. 1-3:
parent PB32(SEQ ID NO. 1);
SPL-12(SEQ ID NO.2) has introduced S76D, P129E, S130K, G260H on the basis of parent;
SPL-17(SEQ ID NO.3) has introduced P129E, S130A, A166D on the basis of parent.
Example 2SPL-12 and SPL-17 Properties improvement compared to the parent PB92
Basic enzymological property detection shows that: as shown in figure 1, SPL-12 and SPL-17 have approximate high pH adaptability with parent PB92, the optimum pH is about 8.0-9.0, and at pH11.0, the residual enzyme activities of parent PB92, SPL-12 and SPL-17 can be still maintained at about 80%; as shown in figure 2, the optimal temperatures of the three are 40 ℃, and the enzyme activity-temperature curves of the SPL-121 and the SPL-17 are better in low-temperature adaptability (the enzyme activity-temperature curves are shifted to low temperature) compared with the parent PB 92.
Parental PB92, SPL-12, and SPL-17 were formulated as 0.5mg/mL solutions using PBS pH 8.0. Samples were taken every 30 min/60 min at 30 and 50 ℃ for enzyme activity, and the results are shown in tables 1 and 2:
table 1: comparison of residual enzyme activity-time of SPL-12 and SPL-17 with that of the parent PB92 (30 ℃ C.)
| Time (min) | Parent PB92 | SPL-12 | SPL-17 |
| 30 | 75.2％ | 85.2％ | 79.2％ |
| 60 | 63.3％ | 77.8％ | 66.9％ |
| 90 | 58.4％ | 68.5％ | 60.4％ |
| 120 | 50.2％ | 60.1％ | 51.2％ |
| 150 | 44.5％ | 55.3％ | 44.9％ |
| 180 | 38.9％ | 50.5％ | 41.3％ |
| 240 | 27.5％ | 48.8％ | 39.8％ |
| 300 | 17.3％ | 43.2％ | 26.0％ |
| 360 | <5％ | 39.6％ | 17.5％ |
Table 2: comparison of residual enzyme activity-time of SPL-12 and SPL-17 with that of the parent PB92 (50 degree centigrade)
The results show that the stability of SPL-12 and SPL-17 is obviously improved at higher temperature. At the general temperature (20-30 ℃) of sludge treatment, the stability of SPL-12 and SPL-17, especially SPL-12 is obviously prolonged, nearly 40% of enzyme activity can still remain after 6 hours, and the method has potential for solving the problem of short enzyme action time in sludge treatment.
Example 3 sludge solubilization experiment Using Complex enzyme preparation
The alkaline protease (parent PB92/SPL-12), lysozyme and cellulase in the prepared complex enzyme are addedAre respectively 2X 106U/g sludge dry weight, 1X 106U/g sludge dry weight, 1X 104U/g sludge dry weight. For the sludge solubilization experiments, the results are shown in tables 3 and 4:
TABLE 3 protein dissolution Effect of Complex enzyme preparation (calculated protein content, mg/L)
It can be seen that SPL-12 with enhanced stability at low temperature effectively improves the protein dissolution effect compared with the parent PB 92: especially after 2 hours, the protein content of the parent group PB92 increased slowly or even stagnated, while SPL-12 still increased considerably (about 30%).
TABLE 4 polysaccharide dissolution Effect of Complex enzyme preparation (calculated sugar content, mg/L)
The sugar elution tendency is similar to the protein elution tendency, and although the effect of SPL-12 compared with the parent PB92 is not different from that of the protein elution experiment, the improvement of the protease stability is also proved to be helpful for the elution of polysaccharide.
Sequence listing
<110> Linxiaoli
<120> alkaline protease low-temperature mutant and application thereof in sludge treatment
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 269
<212> PRT
<213> Alkaliphilic Bacillus (Bacillus alcalophilus)
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1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
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Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
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Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
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His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu
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Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
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Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
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Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
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Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
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Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
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Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
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Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
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Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
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Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
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Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile
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Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
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Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
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<210> 2
<211> 269
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala
1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Asp Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
115 120 125
Glu Lys Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile
225 230 235 240
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255
Tyr Gly Ser His Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265
<210> 3
<211> 269
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala
1 5 10 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser
35 40 45
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr
50 55 60
His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu
65 70 75 80
Gly Val Ala Pro Asn Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala
85 90 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala
100 105 110
Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser
115 120 125
Glu Ala Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser
145 150 155 160
Tyr Pro Ala Arg Tyr Asp Asn Ala Met Ala Val Gly Ala Thr Asp Gln
165 170 175
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile
180 185 190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr
195 200 205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala
210 215 220
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile
225 230 235 240
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu
245 250 255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg
260 265
Claims (3)
1. An alkaline serine protease variant, characterized in that the amino acid sequence of said alkaline serine protease variant is SEQ ID No. 2.
2. Use of an alkaline serine protease variant according to claim 1 in sludge treatment; the sludge is alkaline sludge.
3. Use according to claim 2, wherein the sludge treatment is fermentative acidogenesis.
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| DE102007011236A1 (en) * | 2007-03-06 | 2008-09-11 | Henkel Ag & Co. Kgaa | Carboxyl-bearing benzophenone or benzoic acid anilide derivatives as enzyme stabilizers |
| CN101245327B (en) * | 2008-03-14 | 2010-06-02 | 广东省生态环境与土壤研究所 | Bacillus licheniformis and method for producing alkali proteinase with sewage sludge as raw material |
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