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\begin{document}

\title{Construction, expression and application of microbial \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase
for \selectlanguage{greek}β-\selectlanguage{english}1,3-glucan oligosaccharide production\selectlanguage{english}}



\author[1]{Ying Xu}%
\author[1]{Guoshuai Yang}%
\author[1]{Xiao-Bei Zhan}%
\author[2]{Xiuyu Hu}%
\author[1]{Minjie Gao}%
\affil[1]{Jiangnan University}%
\affil[2]{China Biotech Fermentation Industry Association}%


\vspace{-1em}



  
  \date{May 20, 2024}


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\selectlanguage{english}
\begin{abstract}
\selectlanguage{greek}β-\selectlanguage{english}1,3-Glucan oligosaccharides produced by enzymatic hydrolysis of
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucans are of great interest, because they possess biological
functionalities such as regulating intestinal health, improving immunity
and anti-tumor. A significant qualification for the industrial
production of glucan oligosaccharides is \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase with high
catalytic ability. These enzymes are widely present in fungi, bacteria,
plants and invertebrates, they are separated into glycoside hydrolase
families 3, 5, 16, 17, 55, 64, 72, 81, 128 and 132. This report presents
the research progress on the construction and expression of
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases in Pichia pastoris and Escherichia coli from different
microorganisms. Culture conditions and strategies to improve enzyme
activity in these hosts are summarized. The application of
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases in different \selectlanguage{greek}β-\selectlanguage{english}1,3-glucans (curdlan, scleroglucan,
laminarin, and schizophyllan) degradation and their hydrolysate analysis
are reviewed. This review provides an important reference for further
modification and utilization of microbial \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases in
large-scale production of multi-functional oligosaccharides.%
\end{abstract}%



\sloppy


May 14, 2022

To,

Editor-in-Chief

Biotechnology Journal

Subject: Manuscript submission

Dear Prof. Alois Jungbauer:

Please consider our manuscript entitled ``\selectlanguage{greek}\textbf{Construction,
expression and application of microbial β-1,3-glucanase for β-1,3-glucan
oligosaccharide production}\selectlanguage{english} '' for publication as a Review Article
in\textbf{Biotechnology Journal} \emph{.}

\selectlanguage{greek}β-\selectlanguage{english}1,3-Glucan oligosaccharides produced by enzymatic hydrolysis of
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucans have attracted great interest, because they possess
biological functionalities such as disease-resistance in plants,
immuno-modulating activities in animals and anti-tumor properties.
\selectlanguage{greek}β-\selectlanguage{english}1,3-Glucanase with high specific activity is a prerequisite for the
industrial preparation of glucan oligosaccharides. These enzymes are
widely distributed in bacteria, fungi, plants and invertebrates, and are
classified into glycoside hydrolase families 3, 5, 16, 17, 55, 64, 72,
81, 128 and 132. Different endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases produce various
hydrolysates with different degrees of polymerization, which can be used
not only to prepare oligosaccharides with multiple biological functions,
but also to understand chemical structures and biological functions of
polysaccharides.

The early research of our team focused on the fermentation condition
optimization for the production of \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase by
wild-type\emph{Trichoderma harzianum} GIM 3.442. Coupled fermentation
systems of\emph{T. harzianum} GIM 3.442 and \emph{Agrobacterium} sp.
ATCC 31749/\emph{Sclerotium rolfsii} WSH-G01/\emph{Schizophyllum
commune}GDMCC 5.43 were established for producing linear and branched
\selectlanguage{greek}β-\selectlanguage{english}1,3-glucooligosaccharides. Then, endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase gene (\emph{BGN}
) from \emph{T. harzianum} was expressed in \emph{P. pastoris} GS115
with promoter optimization (pGAP), and a mutant endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase was
obtained by implementing error-prone PCR technology, and the specific
activity towards curdlan was much higher than the original enzyme (292
U/mg) and other reported enzymes, such as recombinant Blg32 from
\emph{Bacillus lehensis} (233.01 U/mg) and Endo23 from \emph{Trichoderma
teesei} GIMCC 3.498(1.52 U/mg). One-step production of functional
branched glucan oligosaccharides was established with coupled
fermentation of \emph{P. pastoris} and \emph{S. rolfsii} , the maximum
yield of \selectlanguage{greek}β- \selectlanguage{english}glucan oligosaccharides (DP 2--17) was 12.71 g/L in a 7-L
bioreactor.

In order to improve the endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase catalytic efficiency and
expression level, great efforts have been made to develop effective
enzyme engineering and fermentation strategies. Numerous genes encoding
endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases have been isolated and characterized from various
organisms, and can be produced in high yields by recombinant expression
methods. This report presents the research progress on the construction
and expression of \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase in \emph{Escherichia coli} and\emph{P.
pastoris} from different microorganisms. Culture conditions and
strategies to improve enzyme activity in these hosts are summarized. The
application of \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase in degradation of different \selectlanguage{greek}β-\selectlanguage{english}1,3-glucans
(curdlan, scleroglucan, laminarin, and schizophyllan) and their
hydrolysate analysis are reviewed. It also summarizes the structural
differences and healthy functional activities of different types of
oligosaccharides. This study provides an important reference for further
modification and utilization of microbial \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanases in
large-scale production of multi-functional oligosaccharides.

I, as the corresponding author, on behalf of all co-authors of the paper
declare that this manuscript has not been previously published and is
not currently submitted for review to any other journal. All authors
declare no conflict of interest. We would greatly appreciate your kind
attention on this submission and look forward to the helpful comments
from you and other referees.

Please let us know if we can provide any additional information to
facilitate your assessment of the suitability of our work for
publication in \emph{BTJ} .

Sincerely yours,

Minjie Gao

\textbf{The list of SCI journals papers published by the corresponding
and first authors}

\begin{enumerate}
\tightlist
\item
  Gao, M. J., Xu, Y., Yang, G. S., Jin, S. X., Hu, X. Y., Jiang, Y.,
  Zhu, L., Li, Z. T., \& Zhan, X. B. (2021). One-step production of
  functional branched oligoglucosides with coupled fermentation
  of\emph{Pichia pastoris} GS115 and \emph{Sclerotium rolfsii}
  WSH-G01.\emph{\textbf{Bioresource Technology}, 335} , 125286.
\item
  Gao, M. J., Yan, J. J., Zhao, Y., Zhu, L., Yang, G. S., \& Zhan, X. B.
  (2021). Expression of a thermostable \selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase
  from\emph{Trichoderma harzianum} in \emph{Pichia pastoris} and use in
  oligoglucosides hydrolysis. \emph{\textbf{Process Biochemistry}, 107}
  , 74--82.
\item
  Gao, M. J., Yang, G. S., Li, F. F., Wang, Z. C., Hu, X. Y, Jiang, Y.,
  Yan, J. J, Li, Z. T., \& Zhan, X. B. (2021). Efficient
  endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase expression in \emph{Pichia pastoris} for
  co-culture with \emph{Agrobacterium} sp. for direct curdlan
  oligosaccharide production. \emph{\textbf{International Journal of
  Biological Macromolecules,} 182} , 1611--1617.
\item
  Gao, M. J., Liu, L. P., Li, S., Lyu, J. L, Jiang, Y., Zhu, L., Zhan,
  X. B., \& Zheng, Z. Y. (2020). Multi-stage glucose/pachymaran
  co-feeding enhanced endo-\selectlanguage{greek}β-\selectlanguage{english}1,3-glucanase production
  by\emph{Trichoderma harzianum} via simultaneous increases in cell
  concentration and inductive effect. \emph{\textbf{Bioprocess and
  Biosystems Engineering,} 43 (8)} , 1479--1486.
\item
  Gao, M. J., Duan, F. Y., Liu, L. P., Hu, X. Y., Zhu, L., Jiang, Y., \&
  Zhan, X. B. (2021). An innovative strategy of recycling miscellaneous
  waste carbohydrates from high-fructose syrup production
  for\emph{Pichia pastoris} fermentation. \emph{\textbf{Journal of
  Cleaner Production,} 326} , 129404.
\item
  Gao, M. J., Li, H. Y., Yang, T. Y., Li, Z. T., Hu, X. Y., Wang, Z. C,
  Jiang, Y., Zhu, L., \& Zhan, X. B. (2022). Production of prebiotic
  gellan oligosaccharides based on the irradiation treatment and acid
  hydrolysis of gellan gum. \emph{\textbf{Carbohydrate Polymers}, 279} ,
  119007.
\item
  Gao, M. J., Zhou, Y. C., Yan, J. J., Zhu, L., Li, Z. T., Hu, X. Y., \&
  Zhan, X. B. (2022). Efficient precious metal Rh(III) adsorption by
  waste \emph{P. pastoris} and \emph{P. pastoris} surface display from
  high-density culture. \emph{\textbf{Journal of Hazardous Materials},
  427} , 128140.
\item
  Gao, M. J., Liu, Z. L., Zhao, Z. S., Wang, Z. C., Hu, X. Y., Jiang,
  Y., Yan, J. J., Li, Z. T., Zheng, Z. Z., \& Zhan, X.B. (2022).
  Exopolysaccharide synthesis repressor genes (\emph{exo} R
  and\emph{exo} X) related to curdlan biosynthesis by
  \emph{Agrobacterium}sp. \emph{\textbf{International Journal of
  Biological Macromolecules,}205} , 193--202.
\item
  Li, Z. T., Hu, G. A., Zhu, L., Zhao, Z. C., Jiang, Y., Gao, M. J., \&
  Zhan, X. B. (2021). In vitro digestion and fecal fermentation of
  highly resistant starch rice and its effect on the gut
  microbiota.\emph{\textbf{Food Chemistry}, 361} , 130095.
\item
  Li, Z. T., Hu, G. A., Zhu, L., Sun, Z. L., Jiang, Y., Gao, M. J., \&
  Zhan, X. B. (2021). Study of growth, metabolism, and morphology
  of\emph{Akkermansia muciniphila} with an \emph{in vitro} advanced
  bionic intestinal reactor. \emph{\textbf{BMC Microbiology}, 21 (1)} ,
  61.
\item
  Li, Z. T., Zhu, L., Hu, G. A., Sun, Z. L., Zhan, X. B., \& Gao, M. J.
  (2021). \emph{Akkermansia muciniphila} fermentation culture based on a
  novel bionic large intestine dynamic digestion
  model.\emph{\textbf{Food Bioscience,} 12 (34)} , 122--127.
\item
  Liang, Y., Zhu, L., Ding, H., Gao, M. J., Zheng, Z. Y., Wu, J. R, \&
  Zhan, X. B. (2017). Enhanced production of curdlan by coupled
  fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma
  harzianum GIM 3.442. \emph{\textbf{Carbohydrate Polymers,} 157} ,
  1687--1694.
\item
  Liang, Y., Zhu, L., Gao, M. J., Wu, J. R., \& Zhan, X. B. (2018).
  Effective production of biologically active water-soluble \selectlanguage{greek}β-\selectlanguage{english}1,3-glucan
  by a coupled system of \emph{Agrobacterium} sp. and \emph{Trichoderma
  harzianum} . \emph{\textbf{Preparative Biochemistry \& Biotechnology},
  48 (5)} , 446--456.
\end{enumerate}

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