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CN-120843483-B - Acid endoglucanase variant and application thereof

CN120843483BCN 120843483 BCN120843483 BCN 120843483BCN-120843483-B

Abstract

The invention discloses an acidic endoglucanase variant, which has at least 80% of identity with the amino acid sequence SEQ ID NO. 2 of cellulase EGII and contains a specific acidic mutation site, wherein the amino acid sequence of the variant is selected from SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12. The acid optimized variant of the sequence has higher enzyme activity under the condition of 50 ℃ acid fiber, obviously improves the enzyme activity and the hydrolysis efficiency under the condition of low pH compared with the wild type (EGII), is suitable for an SSF process without pH adjustment, and reduces the production cost of the biofuel.

Inventors

  • XU PENGFEI
  • ZHANG HONG
  • LI FENG
  • XU SHUANG

Assignees

  • 宜昌东阳光生化制药有限公司

Dates

Publication Date
20260508
Application Date
20250704

Claims (6)

  1. 1. An acid endoglucanase variant, characterized in that the amino acid sequence of the acid endoglucanase variant is selected from the group consisting of SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and SEQ ID No. 12.
  2. 2. A nucleic acid encoding at least one variant of an acid endoglucanase according to claim 1, selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 and SEQ ID NO. 11.
  3. 3. A host cell comprising the nucleic acid of claim 2.
  4. 4. The host cell of claim 3, wherein the host cell is E.coli, trichoderma reesei or Pichia pastoris.
  5. 5. An enzyme composition comprising the acid endoglucanase variant of claim 1 and a β -glucosidase enzyme complex.
  6. 6. Use of the acidic endoglucanase variant of claim 1 for hydrolyzing cellulose and/or producing biofuel at a pH of 3.5-5.5.

Description

Acid endoglucanase variant and application thereof Technical Field The invention belongs to the technical field of genetic engineering and enzyme molecular transformation, and particularly relates to an acidic endoglucanase variant and application thereof. Background Cellulase is used as a core component of a biomass conversion system, and the catalytic efficiency of the cellulase directly influences the production cost of the biofuel. Industrial enzymatic hydrolysis processes are typically performed in an acidic environment at pH 4.0-5.0, but natural endoglucanases (e.g. trichoderma reesei EGII) suffer from the drawbacks of significant attenuation of specific activity (< 50% initial activity), insufficient thermostability (50 ℃ half-life <2 h), mismatch of substrate binding domain charge distribution. CN105874066a discloses that the EG2 variant, although improving thermal stability, does not specifically optimize the acidic catalytic site, resulting in low specific activity at pH 4.0, requiring additional pH adjuster to increase process cost. Therefore, it is of great industrial interest to develop endoglucanase variants having high activity and stability under acidic conditions. Disclosure of Invention The invention provides an acidic endoglucanase variant and application thereof, which has high activity and stability under acidic conditions. In order to achieve the above object, the present invention adopts a technical scheme that an acid endoglucanase variant has at least 80% identity with the amino acid sequence SEQ ID NO. 2 of cellulase EGII and contains a specific acid mutation site, wherein the mutation site is selected from a single or a combination of a plurality of P49T、G51S、S64A、T127N、N146T、N146P、S151N、S151Y、I152L、S153G、V171I、T195A、S203T、N223D、 E233A、A260S、T303V、G309D、G309A、S312A、Q320A、N322G、Q336A、I339E、Q340T、 D341Y、M341L、Q345E、I346V、Q347A、Q351A、G363A、S369T、T370N、V372T、T378N、N382S、W384M、T387Q、S388P、S391A、S392A、A395T and K397S. Alternatively, the amino acid sequence of the variant is selected from SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO. 12. The invention also relates to a nucleic acid encoding at least one of the above acid endoglucanase variants. Alternatively, it comprises a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9 or SEQ ID NO 11. The invention also relates to a nucleic acid comprising a nucleic acid selected from the group consisting of the above. The invention also relates to a host cell comprising a nucleic acid selected from the group consisting of the above. The invention also relates to a host cell comprising a vector selected from the group consisting of the above. Alternatively, the host cell is E.coli, trichoderma reesei, or Pichia pastoris. The invention also relates to an enzyme composition which is a complex comprising the variant and beta-glucosidase. The invention also relates to the use of the acid endoglucanase variant for hydrolyzing cellulose, producing biofuel and/or textile at a pH of 3.5-5.5. The invention has the following beneficial effects: The acidic endoglucanase variant provided by the invention has higher enzyme activity under the condition of 50 ℃ acid fiber, compared with a wild type, the specific activity is generally improved by 54% -109%, and the pH tolerance is better. Is particularly suitable for SSF technology without pH adjustment, and reduces the production cost of the biofuel. Drawings FIG. 1 is a schematic diagram of PCBHI-EGII-TCBHI vector. FIG. 2 is a diagram of a PCR-verified nucleic acid electrophoresis gel. FIG. 3 shows a graph of the enzymatic activity of EGII and mutant CMC produced by fermentation of Trichoderma reesei. FIG. 4 shows EGII and mutant temperature tolerance results. FIG. 5 shows the results of EGII and mutant pH tolerance. Detailed Description Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. In the following examples, E.coli was derived from a well-known competent cell DH 5. Alpha. (KTSM L), trichoderma reesei, pET28a vector, restriction enzyme, etc., were obtained from commercial sources. The EGII sequences and the mutant sequences are shown in Table 1, the mutant sequences are obtained through computer protein simulation and Ai evolution learning, and the synthesis of the specific polypeptide sequences SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12 is completed by the Soujin Zhi Biotech Co., ltd. Wherein the amino acid sequences of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12 have the identity of more than or equal to 80% with SEQ ID NO. 2 and contain specific acidic mutation sites. TABLE 1 Methods of producing the endoglucana