合成生物

生物类型	来源分类	几丁质含量
节肢动物	甲壳纲：虾、蟹等	20%～85%
昆虫纲：蝗虫/蝴蝶/蚊/蛾/蝇/蚕等蛹壳中	20%～60%
多足/蛛形纲：马陆、蜈蚣、蜘蛛、蝎子、螨虫等	4%～22%
软体动物	双神经/腹足/掘足/瓣鳃/头足纲：鲍鱼、蜗牛、角贝、牡蛎、乌贼等	3%～26%
环节动物	原环虫/毛足纲：角涡虫、沙蚕、蚯蚓等	20%～38%
原生动物	鞭毛虫/肉足/孢子虫/纤毛虫纲：锥体虫、变形虫、疟原虫、草履虫等	极少
腔肠动物	水螅虫/钵水母/珊瑚虫纲：水螅、筒螅、海月水母、海蜇、霞水母等	3%～30%
海藻	主要是绿藻	少量
真菌	囊菌、担子菌、藻菌等	微量至45%
动物关节	蹄、足的坚硬部分、动物肌肉、骨结合处等	少量

生物类型	来源分类	几丁质含量
节肢动物	甲壳纲：虾、蟹等	20%～85%
昆虫纲：蝗虫/蝴蝶/蚊/蛾/蝇/蚕等蛹壳中	20%～60%
多足/蛛形纲：马陆、蜈蚣、蜘蛛、蝎子、螨虫等	4%～22%
软体动物	双神经/腹足/掘足/瓣鳃/头足纲：鲍鱼、蜗牛、角贝、牡蛎、乌贼等	3%～26%
环节动物	原环虫/毛足纲：角涡虫、沙蚕、蚯蚓等	20%～38%
原生动物	鞭毛虫/肉足/孢子虫/纤毛虫纲：锥体虫、变形虫、疟原虫、草履虫等	极少
腔肠动物	水螅虫/钵水母/珊瑚虫纲：水螅、筒螅、海月水母、海蜇、霞水母等	3%～30%
海藻	主要是绿藻	少量
真菌	囊菌、担子菌、藻菌等	微量至45%
动物关节	蹄、足的坚硬部分、动物肌肉、骨结合处等	少量

酶	活性	酶活	参考文献
RFChiA	持续性外切	6.9 U/mg	[75]
Chit46	内切	9.5 U/mg	[76]
ActChi	持续性外切	3.7 U/mg	[64]
Chi304	内切、外切	ND	[65]
Chit33	内切	2.7 U/mg	[77]
R-SaChiA4	内切	28 U/mg	[74]
CmChi1	内切、外切、N-乙酰氨基葡萄糖苷酶	1.1 U/mg	[78]

酶	活性	酶活	参考文献
RFChiA	持续性外切	6.9 U/mg	[75]
Chit46	内切	9.5 U/mg	[76]
ActChi	持续性外切	3.7 U/mg	[64]
Chi304	内切、外切	ND	[65]
Chit33	内切	2.7 U/mg	[77]
R-SaChiA4	内切	28 U/mg	[74]
CmChi1	内切、外切、N-乙酰氨基葡萄糖苷酶	1.1 U/mg	[78]

来源	酶	底物	产物	寡糖产率/产量	参考文献
Salinivibrio sp. BAO-1801	野生几丁质酶	胶体几丁质	几丁二糖，少量GlcNAc	71.5%	[97]
T. gamsii R1	野生几丁质酶	胶体几丁质	几丁二糖、三糖	11.62 g/L，1.92 g/L	[98]
S. marcescens	SmChiB	胶体几丁质	几丁二糖	2.04 g/L	[83]
堆肥宏基因组	ActChi	粉粒几丁质	几丁二糖	17%	[64]
T. harzianum	rChit46	胶体几丁质	几丁二糖，微量GlcNAc	94.8%	[76]
P. barengoltzii	PbChi70	胶体几丁质	几丁二糖，微量GlcNAc	21.6 g/L，89.5%	[68]
F. johnsoniae UW101	FjChiB	胶体几丁质	几丁二糖、三糖	—	[63]
S. marcescens	rCHI-2	胶体几丁质	几丁二糖，微量GlcNAc	—	[99]
M. thermophila C1	Chi1	溶胀几丁质	几丁二糖，微量GlcNAc	—	[100]
A. fumigatus df347	AfChi28	胶体几丁质	几丁二糖到几丁四糖	—	[101]
A. media CZW001	AmChi	粉粒几丁质	几丁五糖、六糖	—	[102]
B. aryabhattai	BaChiA	粉粒几丁质	几丁二糖到几丁六糖	—	[103]
Corallococcus sp. EGB	CcCti1	胶体几丁质	几丁二糖到几丁六糖	—	[39]

来源	酶	底物	产物	寡糖产率/产量	参考文献
Salinivibrio sp. BAO-1801	野生几丁质酶	胶体几丁质	几丁二糖，少量GlcNAc	71.5%	[97]
T. gamsii R1	野生几丁质酶	胶体几丁质	几丁二糖、三糖	11.62 g/L，1.92 g/L	[98]
S. marcescens	SmChiB	胶体几丁质	几丁二糖	2.04 g/L	[83]
堆肥宏基因组	ActChi	粉粒几丁质	几丁二糖	17%	[64]
T. harzianum	rChit46	胶体几丁质	几丁二糖，微量GlcNAc	94.8%	[76]
P. barengoltzii	PbChi70	胶体几丁质	几丁二糖，微量GlcNAc	21.6 g/L，89.5%	[68]
F. johnsoniae UW101	FjChiB	胶体几丁质	几丁二糖、三糖	—	[63]
S. marcescens	rCHI-2	胶体几丁质	几丁二糖，微量GlcNAc	—	[99]
M. thermophila C1	Chi1	溶胀几丁质	几丁二糖，微量GlcNAc	—	[100]
A. fumigatus df347	AfChi28	胶体几丁质	几丁二糖到几丁四糖	—	[101]
A. media CZW001	AmChi	粉粒几丁质	几丁五糖、六糖	—	[102]
B. aryabhattai	BaChiA	粉粒几丁质	几丁二糖到几丁六糖	—	[103]
Corallococcus sp. EGB	CcCti1	胶体几丁质	几丁二糖到几丁六糖	—	[39]

酶（来源）	底物	GlcNAc浓度/（g/L）	转化率	参考文献
C. meiyuanensis SYBC-H1发酵液	粉粒几丁质	39.3	98%	[111]
微生物发酵处理几丁质	19.2	96%	[112]
超声处理几丁质	2.65	100%	[113]
高压均质小龙虾壳	3.9	—	[114]
有机溶剂预处理几丁质	4.6～7.6	96%	[115]
碱冻融处理几丁质	75	98%	[116]
A. caviae CH129发酵液	胶体几丁质	—	93%	[117]
A. terreus 发酵液	膨胀几丁质	46	92%	[67]
S. proteamaculans NJ303发酵液	高压均质小龙虾壳	3.9	78%	[114]
T. harzianum发酵液	冻干几丁质粉	14	80%	[118]
S. albolongus 发酵液	胶体几丁质	4.4	89%	[119]
ScChiC, ScHEX	粉粒几丁质	9.4	94%	[120]
SaChiA4, SvNag2557	胶体几丁质	8.0	80%	[121]
ChiA, BsNagZ	胶体几丁质	—	88%	[122]
BpChiA, BlNagZ	胶体几丁质	—	64%	[123]
CmChi1	胶体几丁质	9.8	98%	[78]
ChiG	胶体几丁质	—	—	[124]
AMCase	胶体几丁质	1.2	87%	[125]
PbChi70突变体, NAGase	胶体几丁质	—	97%	[126]
PbChi74, NAGase	胶体几丁质	27.8	93%	[127]

酶（来源）	底物	GlcNAc浓度/（g/L）	转化率	参考文献
C. meiyuanensis SYBC-H1发酵液	粉粒几丁质	39.3	98%	[111]
微生物发酵处理几丁质	19.2	96%	[112]
超声处理几丁质	2.65	100%	[113]
高压均质小龙虾壳	3.9	—	[114]
有机溶剂预处理几丁质	4.6～7.6	96%	[115]
碱冻融处理几丁质	75	98%	[116]
A. caviae CH129发酵液	胶体几丁质	—	93%	[117]
A. terreus 发酵液	膨胀几丁质	46	92%	[67]
S. proteamaculans NJ303发酵液	高压均质小龙虾壳	3.9	78%	[114]
T. harzianum发酵液	冻干几丁质粉	14	80%	[118]
S. albolongus 发酵液	胶体几丁质	4.4	89%	[119]
ScChiC, ScHEX	粉粒几丁质	9.4	94%	[120]
SaChiA4, SvNag2557	胶体几丁质	8.0	80%	[121]
ChiA, BsNagZ	胶体几丁质	—	88%	[122]
BpChiA, BlNagZ	胶体几丁质	—	64%	[123]
CmChi1	胶体几丁质	9.8	98%	[78]
ChiG	胶体几丁质	—	—	[124]
AMCase	胶体几丁质	1.2	87%	[125]
PbChi70突变体, NAGase	胶体几丁质	—	97%	[126]
PbChi74, NAGase	胶体几丁质	27.8	93%	[127]

几丁质资源生物降解和高值转化的研究进展

PDF下载

张阿磊 , 魏国光 , 张弛 , 陈磊 , 周奚 , 刘伟 , 陈可泉

合成生物学 | 特约评述 2024,5(6): 1279-1299

收起

合成生物学 | 特约评述 2024, 5(6): 1279-1299

几丁质资源生物降解和高值转化的研究进展

全屏

张阿磊, 魏国光, 张弛, 陈磊, 周奚, 刘伟, 陈可泉

作者信息

南京工业大学生物与制药工程学院，材料化学工程国家重点实验室，江苏南京 211816

张阿磊（1993—），男，副教授，硕士生导师。研究方向为以酶催化技术转化低值含氮大分子（甲壳素、硝化纤维素等）合成高值含氮化学品如材料单体、功能糖等，涉及酶挖掘、酶改造及酶催化过程强化等技术。E-mail：zhangalei@njtech.edu.cn

陈可泉（1982—），男，教授，博士生导师。主要从事材料单体、医药中间体、营养化学品等生物制造研究，研究方向包括：①酶的挖掘、改造与固定化；②细胞工厂的构建与调控；③生物反应过程装备开发与过程强化；④生物基产品制造与工程化。E-mail：kqchen@njtech.edu.cn

Research progress on bio-degradation and valuable bio-conversion of chitinous resources

Alei ZHANG, Guoguang WEI, Chi ZHANG, Lei CHEN, Xi ZHOU, Wei LIU, Kequan CHEN

Affiliations

State Key Laboratory of Materials-Oriented Chemical Engineering，College of Biotechnology and Pharmaceutical Engineering，Nanjing Tech University，Nanjing 211816，Jiangsu，China

出版时间: 2024-12-31 doi: 10.12211/2096-8280.2024-041

文章导航

摘要

收起

几丁质是由N-乙酰氨基葡萄糖（GlcNAc）通过β-1，4-糖苷键构成的高分子聚合物，是地球上储量最丰富的含氮生物质资源，在自然界分布广泛，主要存在于虾蟹外壳、昆虫外骨骼和真菌细胞壁中。由于几丁质含量巨大、可再生，特别是含有珍贵的氮元素，其资源化利用一直受到广泛关注。然而几丁质结构中丰富的氢键作用力与巨大的分子量，赋予了其高结晶度和不溶于水的特性，导致其降解和高值化利用受到挑战，因此常被作为垃圾丢弃或掩埋，污染环境的同时浪费资源。在几丁质降解利用的众多方法中，生物法因过程环保、反应条件温和等优点，在绿色可持续发展的大背景下展现出巨大潜力。本文首先系统介绍了自然界中催化几丁质降解关键酶的来源与分类、催化机制及特性。其次综述了生物法降解几丁质为单糖（GlcNAc和氨基葡萄糖）和寡糖（几丁寡糖和壳寡糖），以及进一步生物转化合成含氮化合物的现状。最后阐述了几丁质生物降解和高值转化过程中所面临的几丁质降解与转化酶活性低、效率差及成本高昂等诸多挑战，展望了发展迅速的合成生物学在几丁质生物转化中的重要作用，这将为几丁质资源的高效生物炼制提供助力。

关键词

几丁质 / 生物降解 / 几丁降解酶 / 生物转化 / 含氮化学品

Abstract

收起

Chitin, a linear homo-polysaccharides composed of N-acetylglucosamine (GlcNAc) through β-1,4-glycosidic bonds, is the richest nitrogen containing biomass resource on earth, with an annual production of 10 billion tonnes. Chitin is widely distributed in nature, mainly found in the shells of shrimps and crabs, the exoskeletons of insects, and the cell walls of fungi. Due to its abundance and renewablity, especially the presence of the valuable nitrogen element, chitin receives widespread attention. However, the abundant hydrogen bonds in the structure of chitin and its huge molecular weight make it highly crystalline and insoluble in water, which leads to challenges in its degradation and high-value utilization. Thus, chitin resource is often discarded as wastes or buried, leading to serious environment issues and wasted resources. Conversion of abundant chitin resources into high value-added chemicals has both environmental and economic significance. Nowadays, the utilization of chitin resources is mainly done by efficient, low-cost chemical method, but causing huge environmental pollution. Compared with chemical method, the biological method shows great potential in the context of green and sustainable development due to the advantages of environmentally friendly process and mild reaction conditions. In this review, the sources and classifications, catalytic mechanisms and properties of key enzymes for chitin degradation are introduced. Secondly, the current status of chitin biodegradation to monosaccharides (GlcNAc and glucosamine) and oligosaccharides (N-acetyl chitooligosaccharides and chitooligosaccharides), and further bio-converted into nitrogen-containing chemicals are reviewed. Although many studies on enzymes involved in chitin degradation and conversion have been carried out with certain achievements, the diversity and complexity of these enzymes, coupled with the low activity and secretory nature and other factors, have hindered the real industrial chitin degradation and conversion. Consequently, the challenges in biodegradation and high-value conversion process of chitin such as low activity of enzyme, poor efficiency and high cost are highlighted. Finally, the important role of rapidly developing synthetic biology technologies in chitin utilization is envisaged, which will aid the efficient bio-refining of chitinous resources.

Key words

chitin / biological degradation / chitinolytic enzyme / biological transformation / nitrogen-containing chemicals

引用本文

张阿磊, 魏国光, 张弛, 陈磊, 周奚, 刘伟, 陈可泉. 几丁质资源生物降解和高值转化的研究进展. 合成生物学, 2024 , 5 (6) : 1279 -1299 . DOI: 10.12211/2096-8280.2024-041

Alei ZHANG, Guoguang WEI, Chi ZHANG, Lei CHEN, Xi ZHOU, Wei LIU, Kequan CHEN. Research progress on bio-degradation and valuable bio-conversion of chitinous resources[J]. Synthetic Biology Journal, 2024 , 5 (6) : 1279 -1299 . DOI: 10.12211/2096-8280.2024-041

正文

收起

随着全球化石原料逐渐枯竭以及炼制过程导致严重的污染问题，利用可再生资源绿色高效合成化学品受到了越来越多关注^［1-2］。几丁质（chitin），又称甲壳素，是由N-乙酰氨基葡萄糖（GlcNAc）单体组成的高分子线性多糖，在自然界含量仅次于纤维素^［3］，每年产生量达100亿吨^［4-5］，主要以α构型存在于虾蟹壳和真菌细胞壁中。我国虾蟹养殖业和真菌发酵产业极其发达，产生了大量虾蟹壳和菌丝体等富含几丁质的下脚料，但由于缺乏高效的炼制手段，通常被作为垃圾丢弃，浪费资源的同时污染环境^［6］。因此，高效、环境友好地转化丰富的几丁质废弃物为高附加值产品，具有减轻环境负担和创造巨大经济价值的双重意义。科研工作者针对几丁质进行了广泛而深入的探索，目前，其应用领域已扩展至医学治疗、农业增产、环保治理、化学工业加工以及食品加工等多个方面，显示出其在跨学科领域的广泛潜力和应用价值。例如几丁质具有生物可降解性、良好的生物相容性和抗菌能力，可用于制作手术线、止血片及膜片等，在医用材料领域备受关注。另外，几丁质具有抗菌、调节肠道环境等生物活性，因此可作为医药保健品，颇受广大消费者青睐。此外，相比仅含碳、氢和氧元素的木质纤维素，几丁质还富含氮元素，可以省去传统合成过程中提供氮供体的步骤，使其成为了合成含氮化学品的绝佳底物，在化工领域表现出巨大的应用潜力^［7］。

然而自然界中几丁质均是以结晶形式存在，相比其他多聚糖，其巨大的分子量及分子内和分子间存在的大量氢键网络，尤其是单元结构中2位碳上的乙酰氨基赋予了其高度结晶、稳定性和不溶于水的特性，导致人体对其吸收差和难以被转化，限制了其在医药、保健品等领域中的应用^［8］。将其降解为溶解性良好的单体（GlcNAc和氨基葡萄糖）和寡聚体（几丁寡糖和壳寡糖）可以充分发挥其抗肿瘤、抗菌等生物活性，同时也可有效提高其向高附加值含氮化学品如N-乙酰神经氨酸、肝素及呋喃胺类化合物等进一步转化的效率^［9-12］。

几丁质的降解方法主要有化学法和生物酶法。传统化学法（目前的商业化生产工艺）是利用高浓度无机酸分解几丁质（解聚同时发生脱乙酰）生成氨基葡萄糖和壳寡糖盐，再经乙酰化合成GlcNAc和几丁寡糖，经济性差，条件苛刻，且产物被认为是非天然的^［13-14］。随着化学技术的发展，涌现出了一些新的化学法，例如采用离子液体、路易斯酸及固体酸等可以实现几丁质的选择性降解与转化，在几丁质资源的高值化利用方面表现出了一定的应用潜力^［15-17］。除此之外，在自然界中，几丁质降解酶（EC 3.2.1.14）是实现几丁质高效降解的酶，可以通过水解作用特异性断裂几丁质及其脱乙酰衍生物壳聚糖的β-1，4糖苷键分别产生GlcNAc、几丁寡糖和氨基葡萄糖、壳寡糖。自然界中细菌、真菌、植物及动物中均可产生几丁质降解酶，其中微生物来源的酶因活性高和性能优异被研究得最多^［18］。相比化学法，生物酶法过程绿色、条件温和及产物生物活性好，是几丁质降解的优选途径，特别是其也可直接作用富含几丁质的废弃物。美国、日本及加拿大等发达国家利用酶法降解几丁质的产业化已非常成熟，但国内相关的基础研究和产业化应用进程却极其缓慢^［19］。主要原因是几丁质降解酶活性低下，难以高效降解几丁质，尤其是面对拥有高结晶度的天然几丁质，需要加入大量酶来提升降解效率，导致酶成本高。近些年，随着合成生物学的快速发展，几丁质生物高效降解和高值转化取得了一系列进展。因此，本文就几丁质资源的高效生物炼制现状和重要突破展开详述，重点综述了几丁质生物降解为单糖（GlcNAc和氨基葡萄糖）和寡糖（几丁寡糖和壳寡糖），以及进一步转化合成N-乙酰神经氨酸、酪氨酸及呋喃胺等高值含氮化合物的进展。

1 几丁质的提取方法与结构特征

收起

几丁质作为自然界中唯一的含氮多聚糖，分布广泛，是真菌细胞壁、昆虫外骨骼、甲壳类动物外壳及线虫卵壳等的主要构成成分之一，含量为3%～85%（表1）^［20］。从结构复杂的几丁质类生物质中提取几丁质是实现其高效利用的第一步。商业几丁质主要是从甲壳类动物外壳如虾蟹壳中提取制得，包括脱矿物质、脱蛋白、脱色等步骤。几丁质的传统提取主要为化学法，具体为酸碱交替处理和氧化脱色步骤。例如伍军等^［21］利用1.5%盐酸和8%氢氧化钠对虾壳进行处理，获得16%的几丁质得率。此法简单高效、周期短、工艺成熟、但过程需要高温、设备腐蚀大、能耗大，且会产生大量酸碱废水。因此，目前绿色化学法如利用一些优良溶剂如离子液体（IL）和深度共熔溶剂（DES）来提取几丁质的研究热度日益增加^［22］。例如，Setoguchi等^［23］利用1-烯丙基-3-甲基咪唑溴化物处理蟹壳，所得几丁质的蛋白含量＜0.1%，脱乙酰程度<7%。Zhu等^［24］利用氯化胆碱-丙二酸制备的DES从龙虾壳中提取几丁质，所得几丁质与商业化几丁质产品的结构相似，纯度较高，提取率高于酸碱法。尽管此法工艺更为绿色，溶剂可重复使用，但IL和DES高昂的成本，且多次使用后溶解的蛋白会导致体系黏稠，在一定程度上限制了其规模化进展。

此外，绿色、温和的生物发酵处理法也得到了广泛关注，主要是利用乳酸杆菌属、芽孢杆菌属、假单胞菌属和沙雷氏菌属等微生物通过单菌或混菌发酵产生的有机酸和蛋白酶来去除虾蟹壳中的矿物质和蛋白，是目前前景较好的一种方法。Sorokulova等^［25］采用生物发酵法提取几丁质，结果表明对废弃虾壳的脱蛋白率和脱矿盐率分别可达到97.1%和92.8%。李磊^［26］利用嗜酸乳酸杆菌发酵处理虾头，72 h蛋白质和矿物质去除率分别为94.8%和0.77%。刘斯雅等^［27］先利用乳酸菌发酵虾壳废弃物，灰分脱除率达到97.5%，再利用解淀粉芽孢杆菌发酵处理，蛋白脱除率可达96.8%，最终两步发酵处理后，几丁质提取率为19.6%。Khanafari等^［28］对比了化学和微生物发酵法从废弃虾壳中提取几丁质，结果表明微生物法较化学法的收率更高，且对几丁质结构影响较小，但纯度低于化学法。

与纤维素（单体为葡萄糖）类似，几丁质的单体是GlcNAc，其2号C位置上为乙酰氨基，相邻的两个GlcNAc反向呈180°排列，并以β-1，4糖苷键连接形成线性链状结构［图1（a）］。根据链的方向差异几丁质可分为α、β及γ型构型^［29-30］，α-几丁质的两条长链以反向平行方式排列^［31］，形成多晶结构，β-几丁质的两条长链以平行方式排列^［32-33］，形成单斜晶结构，γ-几丁质则是α和β型的混合。几丁质的三种晶型（α、β、γ）之间可以通过特定的物理化学手段实现相互转化。具体而言，利用甲酸、盐酸等强酸处理β-几丁质可以转化为α-几丁质；同样，通过饱和硫氰酸锂处理γ-几丁质也能得到α-几丁质。然而，值得注意的是，α-几丁质在现有的条件下难以直接恢复到β-几丁质或γ-几丁质的原始结构。相比β型及γ型，α-几丁质分子内和分子间的氢键网络最为丰富和复杂，赋予了其很高的结晶度和不溶特性，构成了抗降解屏障［图1（b）］。

2 几丁质降解酶

收起

2.1 几丁质降解酶的来源与分类

几丁质降解酶是一类能特异性地断裂GlcNAc-GlcNAc、GlcNAc-GlcN及GlcN-GlcN糖苷键形成可溶性糖的酶的总称，其在自然界中占据重要地位，不仅在碳氮循环、植物免疫、生物防御等生态过程中发挥关键作用，还参与甲壳类动物的蜕壳过程，广泛分布于细菌、真菌等微生物，以及植物和节肢类动物中。其中微生物来源的酶因具有种类多、活力高和性质优异等优点，研究得最多^［34-35］。例如从自然界中筛选得到的Serratia marcescens ^［36］、Aeromonas sp.^［37］、Bacillus aryabhattai ^［34］、Chitinibacter sp. GC72^［38］、Clostridium sp.^［39］、Chitinolyticbacter meiyuanensis SYBC-H1^［40］、Fusarium oxysporum ^［41］、Penicillium aculeatum ^［42］、Thermomyces lanuginosus ^［43］、Trichoderma harzianum ^［44］等微生物均被报道了可以分泌高效的几丁质降解酶。

几丁质降解相关酶主要包括几丁质酶、壳聚糖酶、几丁质脱乙酰酶、N-乙酰氨基葡萄糖苷酶、多糖裂解单加氧酶（lyticpolysaccharide monooxygenase， LPMO）及几丁质结合蛋白（chitin-binding protein， CBP）等。几丁质脱乙酰酶是可以脱除几丁质分子中的乙酰基产生壳聚糖的一类酶^［45］。LPMO是一种新型的铜离子依赖型多糖降解酶，属于辅助类酶，能够以氧化方式打开几丁质链中的糖苷键，形成含有N-乙酰氨基葡萄糖酸的游离末端，为几丁质酶提供更多的酶解位点，从而增加降解效率^［46］。CBP是一类可与几丁质特异结合的蛋白质，可以使几丁质结晶区蓬松化，后来证明其也是一种LPMO^［47］。N-乙酰氨基葡萄糖苷酶的作用是催化可溶性几丁寡糖的β-1，4糖苷键产生GlcNAc，难以降解不溶性几丁质底物^［48］。在几丁质的降解中，几丁质酶和壳聚糖酶是两个关键酶，几丁质酶基于序列相似性主要被归类为糖苷水解酶（GH）18家族和GH19家族^［49］，而壳聚糖酶主要分为GH8和GH46家族^［50］。根据水解模式可以分为外切型和内切型，内切酶可在糖链内部任何位置随机断裂几丁质或壳聚糖，生成聚合度不同的几丁寡糖（聚合度2～7）和壳寡糖（聚合度2～20），但随着时间延长，最终产物的聚合度主要为1～3^［51］。外切酶从糖链的非还原性和还原性末端的外部位点逐渐水解，主要产物为几丁二糖和壳二糖。以几丁质的酶法高效降解为例，主要涉及LPMO、几丁质外切酶、几丁质内切酶和N-乙酰氨基葡萄糖苷酶四种酶的协同作用，对几丁质糖链的具体作用模式如图2所示。

在自然界中，微生物是几丁质资源循环的主要生物，为了高效利用几丁质，一种微生物通常会分泌多种几丁质降解相关酶，从而构成酶系，且相互之间具有较好的协同催化作用。例如，典型几丁质降解细菌S. marcescens能够产3个GH18家族几丁质酶（ChiA、ChiB、ChiC）、1个GH20家族N-乙酰氨基葡糖苷酶及1个LPMO。Chen等^［52］通过基因组学对几丁质高效降解细菌Chitinibacter sp.GC72进行了分析，其含有17个几丁质降解酶，其中12个GH18家族几丁质酶，3个GH19家族几丁质酶、1个GH20家族N-乙酰氨基葡萄糖苷酶和1个AA10家族LPMO。嗜热链霉菌Streptomyces sp.F-3分泌6种几丁质降解酶，包括SsChi18A、SsChi18B、SsChi18C、SsChi19A、SsGH20A和SsLPMO10A^［53］。

2.2 几丁质降解酶挖掘、异源表达及酶学特性

在几丁质降解酶挖掘、异源表达及特性研究方面，印度海得拉巴大学Appa Rao Podile、挪威生命科学大学Vincent G.H. Eijsink、南京工业大学陈可泉、中国农业大学江正强、天津科技大学路福平、中国海洋大学毛相朝、华南理工大学罗晓春、大连理工大学杨青及北京化工大学张桂敏等国内外团队开展了大量且系统的研究。传统的几丁质降解酶获取是通过水解透明圈和颜色等筛选手段，从自然界中特殊环境中筛选分离出产酶活性较高的微生物菌株，再经发酵培养获得^［54-55］，例如南京工业大学陈可泉等^{［38， 56］}通过透明圈筛选，从池塘底泥中获得了两株高效几丁质降解菌Chitinolyticbacter meiyuanensisi SYBC-H1和Chitinibacter sp. GC72，其分泌酶可以高效降解粉粒几丁质和胶体几丁质为GlcNAc。但野生菌的生长性能较差，且产酶能力不稳定，难以满足规模化制备的需求。异源克隆表达是获得大量目的酶的有效手段，而获得高效几丁质降解酶的编码基因是关键。从筛选得到的微生物中克隆相关基因，过程烦琐，且自然环境中存在的微生物资源99%以上难以培养，因此存在一定的盲目性和局限性^［57］。

宏基因组学和基因测序技术的快速发展，为酶的挖掘提供了更为全面和高效的手段，现在可以从EBI metagenomics、iMicrobe、IMG/M等公开数据库下载到大量各种自然环境样品的宏基因组数据，包含了许多与几丁质降解相关的功能基因，并已成为现代酶学研究中重要而珍贵的资料库。目前已经开发出一系列以比对序列为依据或者以家族分析为依据的工具（例如BLAST、FASTA、hmmsearch、PSI-BLAST等），可以在宏基因组数据库中高效检索到大量功能性基因信息。例如，在CAM＿PROJ＿GOS单个宏基因组数据集上使用hmmsearch工具能够找到3400多条几丁质降解酶基因序列。但是如何从庞大的基因数据精准快速获得性能卓越的新基因，这已经成为宏基因组学研究中亟待解决的科学问题^［58］。

获得高效几丁质降解酶后，其高效可溶性表达是其能实际应用的保障。目前大量几丁质降解酶已经在大肠杆菌、毕赤酵母、芽孢杆菌等高效底盘细胞中进行了成功表达。例如，Chen等^［52］通过全基因组测序和粗酶肽指纹图谱图谱分析，从高效几丁质降解细菌C. meiyuanensis SYBC-H1和Chitinolyticbacter sp. GC72中鉴定出了数种几丁质降解关键酶CmChi1、CmChi3、CmChi4、CmNAGase及NAGase159等的编码基因，并实现了在大肠杆菌中的可溶性表达。尽管大肠杆菌的表达量非常出色，然而许多几丁质降解酶在大肠杆菌中表达时会产生无活性的包涵体，而在甲醇诱导下在毕赤酵母中可成功分泌出有活性的酶，如Liu等^［59］报道了来自Fusarium solani 0114 的壳聚糖酶基因在毕赤酵母中表达并实现了大规模发酵生产。因此在不同的几丁质降解酶表达中，常常需要对表达底盘进行筛选。

几丁质降解酶的生产宿主广泛分布于地球生态系统中，它们为适应多样化的环境，进化出了多种性能各异的特性。然而，若要这些酶发挥较佳活性，通常需要在相对温和的环境条件下进行。例如细菌和放线菌来源的几丁质酶在pH 4～7时活性较好^［60］。相对于其他多种宿主，微生物因极端耐受性更强，因此其来源的酶学性能（如pH和温度）更为多样。源自链霉菌的几丁质酶在pH为8～14的范围内仍能保持超过50%的活力，表现出较宽的pH耐受性^［61］。同时，Bacillus subtilis的几丁质酶在高温下具有显著稳定性，能在100 ℃处理20 min后几乎不损失活力^［62-63］。Sun等^［64］从堆肥的宏基因组中挖掘获得了一个嗜热几丁质酶ActChi，其最适温度为80 ℃。Chen等^［65］分离自Aspergillus fumigates的壳聚糖酶在80 ℃和100 ℃条件下半衰期分别达205 h和32 min，展示了优异的热稳定性，在工业应用中具有显著优势。另外，Pseudoalteromonas sp.DL-6生产的几丁质酶ChiA和ChiC在低温下保持高活性，最适温度分别为20 ℃和30 ℃^［66］。此外，Das等^［67］从海洋真菌Aspergillus terreus和Yang等^［68］从Paenicibacillus barengoltzii中分离的几丁质酶，展现出强大的几丁质降解能力，且具有耐高盐高温特性，对含盐量高的海产品废弃物降解具有潜在应用前景。

几丁质降解酶往往表现出严格的底物专一性，例如几丁质酶一般仅催化几丁质类底物如几丁寡糖、胶体几丁质、粉粒几丁质及虾蟹壳等，对壳寡糖、壳聚糖、纤维素等无水解作用。同样壳聚糖酶也展现出水解专一性，优先作用于脱乙酰度（DDA）高的壳聚糖，对几丁质类底物几乎无水解作用或表现出微弱活性。此外，随着壳聚糖DDA的增加，酶的相对活性也相应增强。而N-乙酰氨基葡萄糖苷酶仅能催化低聚几丁寡糖，对几丁质等高分子底物难以作用^［69］。针对同一种酶的不同家族在底物的偏好性方面无明显差异，例如GH18和GH19家族几丁质酶均可降解几丁质，且特异性并不随着家族的变化展现出规律性，少数几丁质酶也对壳聚糖和纤维素表现出一定活性，这多与蛋白序列和高级结构差异有关^［70］。

此外，由于天然几丁质拥有极高的结晶度和分子量，导致大部分几丁质酶对其降解能力较低，因此目前研究多采用物理法（高压均质、超声、球磨等）、化学法（酸溶析、碱冻融、有机溶剂、离子液体等）和生物法（酶法、发酵法）等手段对结晶几丁质进行预处理，可有效提升酶解效率（2～18倍）^［71-73］。虽然预处理可有效降低几丁质结晶度，提高其酶解聚效率，但无疑增加了过程复杂性和成本。挖掘可直接降解天然结晶几丁质的高效酶是攻克此问题的关键。可实现几丁质解聚的酶主要包括持续性几丁质外切酶和几丁质内切酶。其中，持续性外切酶在结晶几丁质解聚中起到最主要的作用，能够剥离出结晶区单条几丁质链并沿其滑动，并在每次催化后不脱离几丁质链而进行持续催化。但目前已报道的可直接解聚结晶几丁质的内切酶和外切酶活力仍不够高（最高分别为28 U/mg和6.9 U/mg）^［74-75］，无法满足实际需要（表2）。

2.3 几丁质降解酶的结构特征和催化机制

鉴于底物分子量的庞大，几丁质降解酶常由多个结构域构成，除核心催化结构域外，还包括如碳水化合物结合模块（carbohydrate binding module， CBM）或Ⅲ型纤维连接蛋白（FnⅢ）等底物识别与结合结构域。几丁质结合结构域（chitin binding domain， ChBD）是几丁质酶中常见的结构域，其能够特异性识别和结合几丁质，促进几丁质酶对几丁质的降解效率^［79-80］。ChBD主要被归属于CBM5、CBM12和CBM18家族，不同来源的几丁质酶上一般具有1～3个ChBD。例如在我们前期研究工作中，在几丁质高效降解菌C. meiyuanensis SYBC-H1中的20多种和Chitinolyticbacter sp. GC72中的10多种几丁质酶大多拥有2个ChBD，说明ChBD在几丁质的降解中起到重要作用^［52］。

不同家族几丁质降解酶的分布广泛且结构差异显著，目前关于不同家族的进化关系尚不明确，可能来源于不同的祖先基因，也有可能是共同祖先，但为了适应不同的生态环境进行了差异性进化。不同家族酶的催化结构特征和催化机制存在一定差异，很多文献和综述已经详细报道，例如GH18家族和GH19家族几丁质酶之间。本文以GH18家族的几丁质酶为案例进行阐述，该家族酶具备典型的（β/α）8-TIM桶状结构，由八个α螺旋和八个β折叠无序卷曲构成，催化裂隙位于TIM桶的上方。GH18几丁质酶进一步细分为A、B、C三个亚家族^［81］。特别地，SubA几丁质酶在Loop7上拥有（α+β）插入结构域（CID），而SubB和SubC则不具备此特征。这些酶的氨基酸序列包含保守的“DXXDXDXE”催化模块和“SXGG”底物结合模块，其中催化模块中的谷氨酸（Glu）和第二个天冬氨酸（Asp）是活性中心［图3（a）］^［82］。相比GH19家族几丁质酶（催化结构域高度螺旋化，其底物结合裂隙浅），GH18几丁质酶底物结合裂隙比较深，与几丁质内切酶不同的是，外切酶的底物结合裂隙“深且窄”，可紧密结合底物，往往表现出持续性降解作用。GH18几丁质酶为底物辅助保留型催化断键机制，以S. marcescens来源的几丁质酶SmChiB（PDB 1E6N）为例，当GlcNAc进入-1亚位点时，该酶通过在氨基酸残基的作用使GlcNAc由椅式变为船式构象，进而促使N-乙酰基的氧原子靠近异头碳（C1）并发起亲核攻击。在此过程中，Tyr214与氧原子形成氢键稳定该结构^［83］。随后，Asp142翻转与N-乙酰基形成氢键，稳定中间体，并接近Glu144形成氢键，激活质子供体，导致糖苷键断裂并形成&#x2bac7;唑啉离子中间体。接着，水分子对C1进行亲核攻击，使&#x2bac7;唑啉离子中间体解体。之后，Glu144恢复初始状态，而Asp142在Asp140的辅助下翻转回原位，完成水解过程［图3（b）］。

此外，对GH18 SubA家族几丁质外切酶的持续性作用机制也已有些了解，例如Nakamura等^［84］采用原子力显微镜单分子成像、X射线衍射和原子动力学模拟等手段，深入研究了SmChiA（PDB 1EHN）的单向持续性运动机制，其运动过程被划分为化学步骤（涵盖底物辅助催化与产物释放）和机械步骤（涉及脱晶与链滑动）。在分子结构水平，持续性外切酶活性架构中的芳香族氨基酸构成了长且深的底物结合裂隙，Loop7上特有的（α+β）CID更加拓宽了底物结合裂隙，使糖链结合亚位点扩展为8个（-6到+2），形成了一个灵活的鞘，推动几丁质链沿其滑动。此外，活性架构中极性残基与芳香残基均呈线性排列［图3（c）］，也可能与酶的持续性有关。

此外，几丁质降解酶的产物聚合度有一定的差异，例如几丁质酶和壳聚糖酶以聚合度（DP）2～3的寡糖产物为主，部分酶也会产生高DP的寡糖，目前关于产物DP的调控机理尚不明确。产物DP受多种因素影响，例如酶本身的特异性和活性，不同类型的酶对糖链的特定部位具有不同的作用特异性，一些酶可能在DP较高的链上表现更为活跃，而另一些可能更倾向于作用于短链或链的末端，同时部分几丁质降解酶表现出转糖基和逆水解活性，可以在较短时间内催化低DP的寡糖产生较高DP的寡糖^［42］。同时，反应条件、底物几丁质的类型等也会影响产物DP，例如酶与糖链作用的时间长短会影响降解深度和程度，进而影响产物的DP。

2.4 几丁质降解酶的蛋白质工程改造

为满足工业应用中的成本需求，提高几丁质降解酶的催化活性和热稳定性已成为酶设计领域的重点研究方向。传统的研发方法倾向于借助定向进化技术，该技术通过随机诱变或基因重组的策略获得突变体，这一过程无需预先对酶的结构和功能进行详尽的分析。例如，Songsiriritthigul等^［85］利用定向进化技术，对源自Bacillus licheniformis的GH18家族ChiA酶进行了易错突变处理，成功筛选出了一个酶活性相较于原始酶提高了2.7倍的突变体。Menghiu等^［86］利用易错PCR方法构建了一个B. licheniformis几丁质酶ChiA的突变体库，以4-甲基伞形酮衍生化的几丁三糖为底物（几丁质酶水解时会释放游离的4-甲基伞形酮，其在365 nm激发时产生荧光），随后运用荧光激活细胞分选（FACS）技术测定突变体的荧光信号强弱，从该库中筛选出了一个酶活性显著增强的突变体DH08。潘梦妍等^［87］通过利用易错PCR技术，成功构建了一个包含几丁质酶Chisb的随机突变文库，以此实现对其的定向进化。随后，采用96孔板进行初步筛选，再辅以摇瓶复筛的方式，进一步确认了酶催化效率的提升，最终获得了两个催化效率显著提高的突变体C43D和E336R。但是定向进化过程中构建的突变文库容量大，需要建立高通量筛选方法。

随着蛋白解析技术和计算机建模技术的日益发展，获得大量精准的蛋白结构，基于结构基础的半理性设计和理性设计策略得到研究者大量关注，其针对特定结构域内的残基进行突变，构建出精简而高效的突变体库。例如，基于共识的半理性设计方法被应用于几丁质酶PpChi1中，通过引入二硫键和脯氨酸的改造策略，显著提升该酶的热稳定性。在50 ℃的条件下，改造后的几丁质酶PpChi1的半衰期达到了野生型酶的26.3倍，这一成果充分展示了结构域内残基突变在提高酶稳定性方面的巨大潜力^［88］。对S. marcescens几丁质酶ChiB的表面残基G188A/A234P进行突变，其在57 ℃时半衰期增加了10倍，T _m增加了4.2 ℃^［89］。Zhao等^［90］对几丁质酶SsChi18A进行了半理性改造后，突变体Y286W、E287A、K186A对胶体几丁质的活性较野生型分别提高了151%、135%和129%。除了催化域的改造，对几丁质降解酶的底物结合域进行替换也可有效提高其降解活性。例如，用来Bacillus circulans WL-12几丁质酶BcChiA1的CBM12代替Streptomyces albolongus ATCC 27414几丁质酶SaChiA4的CBM5，使其对几丁质粉粒的活性提高了54%^［91］。Wan等^［64］将嗜热几丁质酶ActChi的malectin和FnⅢ结构域替换为ChBD，使其对结晶几丁质的降解效率提升4倍。这些策略使几丁质降解酶的稳定性和活性等性能更加优异，对其实际应用具有重要意义。

3 几丁质生物降解与高值转化

收起

3.1 几丁质生物降解合成几丁寡糖和壳寡糖

几丁寡糖［聚合度（DP）2～7］和壳寡糖（DP 2～20）作为水溶性功能低聚糖，拥有抗肿瘤、抗炎和抗菌等优良生物活性，在生物医药、食品、农业等领域具有巨大的应用前景，其市场需求量逐渐增加。酶法降解几丁质及其脱乙酰产物壳聚糖因过程绿色、产品生物活性好及产物聚合度集中等优势，是实现几丁寡糖和壳寡糖制备的优选途径。目前利用酶法降解壳聚糖制备壳寡糖产业化已经非常成熟，例如，Doan等^［92］利用Paenibacillus sp. TKU047来源内切壳聚糖酶TKU047粗酶降解脱乙酰度为98%的壳聚糖，产物为DP 2～9的壳寡糖，产率为68.44%。罗洒等^［93］克隆表达了来自Bacillus amyloliquefaciens ECU08的内切壳聚糖酶，该酶降解底物后主要产生DP 2～3的壳寡糖，在30 L的反应体系中总产物收率高达75.8%，其收益较高，适用于大规模生产。Zhou等^［94］在毕赤酵母中表达的来自烟曲霉的壳聚糖酶Csn75与多数内切型壳聚糖酶不同，该酶在制备高DP壳寡糖方面显示出巨大潜力。为获得特定聚合度的壳寡糖，通常需运用蛋白质工程和酶膜反应器等方法精确控制水解工艺^［95］。例如Kuroiwa等^［96］利用固定化酶填充床反应器连续生产壳五糖和壳六糖，可以连续反应一个月，提高产率的同时可以有效地控制反应的终止，对于制备高聚合的壳聚糖有重要价值。但目前从几丁质制备壳聚糖是采用高温浓碱法，存在较大的污染问题。利用脱乙酰酶对几丁质进行脱乙酰制备壳聚糖具有一定前景，但活性和效率低下，目前仅仅停留在实验室阶段，尚无法进行规模化应用。

相比酶法降解壳聚糖制备壳寡糖，酶法降解几丁质制备几丁寡糖的基础研究和工业化进程却极其缓慢。国内仅有青岛博智汇力生物科技有限公司有少量DP 2～7的几丁寡糖标准品售卖，价格极高（人民币1000～3000元/10 mg）。目前报道中几丁质酶的降解产物主要为几丁二糖和几丁三糖，且常常伴有少量GlcNAc，难以获得高DP的产物（表3）。

例如，Le等^［97］利用来源Salinivibrio sp. BAO-1801几丁质酶水解胶体几丁质，在反应进行8 h后，产物主要为几丁二糖，伴有微量GlcNAc，二糖产率为71.5%。Wang等^［98］从虾壳废弃物中筛选到产低温几丁质酶的菌株Trichoderma gamsii R1，利用纯化的几丁质酶水解胶体几丁质生成11.62 mg/mL几丁二糖和1.92 mg/mL几丁三糖。Wan等^［64］从堆肥宏基因组中挖掘到了一个嗜热几丁质酶ActChi，并经过ChBD的融合构建了人工酶，利用其降解几丁质，产物仅有几丁二糖。从上述案例中发现，酶法降解几丁质制备几丁寡糖在效率上仍然较低，且产物主要的几丁二糖，需要开发出可合成高聚合度几丁寡糖的高效酶。目前仅有少数几丁质酶降解几丁质时可以产生高聚合度几丁寡糖。例如Luo等^［104］利用融合几丁质酶Chit46-CBM3降解几丁质和虾壳废弃物，可以产生DP 2～7的几丁寡糖，产量为8.8 g/L。吴敬等^［105］利用Aeromonas veronii来源的几丁质酶ChiB565水解几丁质主要产生几丁二糖、三糖和四糖，伴有少量几丁五糖和六糖。Raval等^［34］利用Bacillus aryabhattai几丁质酶降解几丁质粉粒合成了DP 2～7的几丁寡糖。此外，少数几丁质酶和N-乙酰氨基葡萄糖酶（NAGase）在发挥水解活性的同时，常伴有转糖苷活性，具有合成更高聚合度寡糖的潜力。例如，印度海得拉巴大学Podile教授团队在几丁质酶的转糖苷活性方面开展了大量研究工作，他们利用Chitiniphilus shinanonensis来源的CsChiL催化几丁四糖合成了几丁六糖^［106］，Flavobacterium johnsoniae来源的ChiA以几丁四糖合成了几丁五糖和几丁六糖^［63］。Singh等^［107］利用Aspergillus oryzae来源的GH20家族NAGase能以几丁二糖、几丁三糖、几丁四糖分别合成聚合度2～4、4～6、5～6的几丁寡糖。Stenotrophomonas maltophilia来源的GH20家族NAGase可分别催化几丁二糖和三糖分别合成了几丁三糖和几丁四糖。Zhang等^［108］发现GH20家族CmNAGase能水解催化DP 2～6几丁寡糖，同时可依次加上1分子GlcNAc生成DP为3～7的几丁寡糖，此外也能以GlcNAc为底物合成几丁二糖。但这些酶的水解活性大于转糖苷和逆水解活性，随着长时间的反应，高聚合度产物仍会被降解，难以积累。

除了专一性几丁质酶和壳聚糖酶，非专一性酶如溶菌酶、胃蛋白酶、脂肪酶、纤维素酶也可用于降解几丁质或壳聚糖制备寡糖。例如，黄晓月等^［109］以被广泛用于食品工业的木瓜蛋白酶作为催化剂水解壳聚糖，最终获得DP 6～10的壳寡糖，产率为45.07%。董惠忠^［110］设计了一个由纤维素酶、木瓜蛋白酶以及壳聚糖酶组成的复合酶系统，用于水解壳聚糖。通过此系统，高效地制备了DP 6～8的壳寡糖，且其产率高达79.84%。这一创新方法显著提高了壳寡糖的制备效率和产物的纯度，为壳寡糖的应用研究提供了有力的技术支持。

3.2 几丁质生物降解合成N-乙酰氨基葡萄糖和氨基葡萄糖

N-乙酰氨基葡萄糖（GlcNAc）是几丁质的基本组成单元，具有修复软骨组织、抗菌抗氧化及保水保湿等众多功能，同时因含有珍贵的氮元素，也是合成神经氨酸、透明质酸及呋喃环类化合物等重要含氮分子的重要前体，在医药、化妆品与保健品等领域受到广泛关注。将几丁质彻底转为GlcNAc往往需要至少2个酶的联合协同作用，内切酶或外切酶将几丁质水解为几丁寡糖，进一步在N-乙酰氨基葡萄糖苷酶NAGase的作用下转化为GlcNAc。迄今为止，已经有诸多利用几丁质酶降解几丁质生产GlcNAc的报道（表4）。

例如，Chen等^［52］在降解几丁质制备GlcNAc方面开展了大量工作，利用C. meiyuanensis SYBC-H1粗酶液对几丁质进行了高效降解，基于酶对底物几丁质的特异性亲和吸附性能，建立了新颖的酶亲和吸附分离与降解的耦合过程，单批酶解几丁质产GlcNAc的浓度可达40 g/L，且酶可重复使用3次，构建了高效的降解工艺。通过C. meiyuanensis SYBC-H1几丁酶粗酶降解碱冻融处理的几丁质，GlcNAc浓度达75 g/L^［116］。此外，此研究团队开发了高压均质^［114］、细菌发酵^［112］、有机溶剂溶析^［115］、超声^［113］、球磨^［111］等系列几丁质预处理工艺，有效提升了酶法制备GlcNAc的效率^［112］。其他团队在GlcNAc的酶法制备上也取得了良好进展，Suresh等^［128］将Aspergillus flavus来源的N-乙酰氨基葡萄糖苷酶降解虾壳来源几丁质生产GlcNAc，产量达到了21.37 g/L。Yang等^［129］利用三个细菌几丁质酶SmChiA、SmChiB、SmChiC和一个昆虫NAGase OfHex1降解粉粒几丁质，24 h产生2.7 g/L GlcNAc（占比95%）。Jiang等^［68］利用细菌几丁质酶PbChi70突变体和真菌NAGase降解粉粒几丁质，24 h产生7.19 g/L的GlcNAc（占比95%），转化率72%。Han等^［126］通过将P. barengoltzii几丁质酶PbChi70突变体和NAGase在黑曲霉进行双酶共表达，在5 L发酵罐中实现了高密度发酵产酶，几丁质酶和NAGase活性最高分别可达61.33 U/mL和353.1 U/mL，利用双酶降解粉状几丁质生产GlcNAc，其纯度≥95%（质量分数），转化率可达71.9%（质量分数）。几丁质酶也可直接降解几丁质富含物，例如Zhang等^［114］使用高压均质处理小龙虾壳，提高了几丁质酶对其的降解效率。Gao等^［130］分离了一株新的几丁质降解菌Chitinibacter sp. GC72，并利用其几丁质酶降解100 g小龙虾壳粉，可得到15.2 g的GlcNAc。此外，自然界中存在少数多功能几丁质酶，可以单酶降解几丁质为GlcNAc，例如Okazaki等^［124］利用C. shinanonensis SAY3^T几丁质内切酶ChiG可以水解胶体几丁质为单一GlcNAc。Chen等^［78］利用C. meiyuanensis SYBC-H1的几丁质酶CmChi1兼具内切、外切及N-乙酰氨基葡萄糖苷酶NAGase活性，可以水解胶体几丁质为GlcNAc，浓度达9.8 g/L，转化率接近100%。但多功能活性中NAGase活性较差，若通过酶催化机制解析和蛋白质工程改造提高多活性的协调性，将为低成本、高效降解几丁质制备GlcNAc提供可能。

氨基葡萄糖（GlcN）是GlcNAc的脱乙酰产物，传统的合成方法是通过酸处理对几丁质进行水解。酶法是通过N-乙酰葡糖胺脱乙酰酶催化生成GlcN，但效率低下，因此研究较少。例如，从海洋环状杆菌中分离出一种脱乙酰酶，其可催化GlcNAc转化为GlcN，经过72 h后，可从几丁质和蘑菇中分别得到18 mg/g和7 mg/g的GlcN^［131］。

3.3 几丁质生物合成含氮化学品

除了降解合成单糖和寡糖，几丁质也可以通过生物转化合成生物能源。例如毛霉能够将几丁质粉末转化为生物燃料乙醇^［132］。细菌Shewanella oneidensis可以通过微生物燃料电池系统将几丁质转化为电能^［133］。此外，相较于纤维素仅含碳、氢、氧元素，几丁质含有珍贵的氮元素，因此也是合成含氮化合物的绝佳底物，可以省去氮元素的额外供给。例如，Liu等^［134］构建了几丁质酶、N-乙酰氨基葡萄糖-2-差向异构酶和N-神经氨酸醛缩酶的三酶级联催化体系，在24 h内可以催化20 g/L胶体几丁质获得9.2 g/L的N-乙酰神经氨酸。Ma等^［135］开发了一种综合生物精炼工艺将虾蟹壳废弃物来源的几丁质转化为两种芳香族含氮化学品，首先利用酶降解几丁质，再利用工程大肠杆菌以几丁质降解物发酵合成了0.91 g/L酪氨酸和0.41 g/L左旋多巴。

近年来以几丁质为底物出发合成呋喃环类含氮化合物也取得了相当大的进展（图4）。由于这些化合物在生物体内没有天然的合成和代谢路径，目前主要经过生物和化学催化结合的方式合成。其中的关键步骤是几丁质单体GlcNAc脱水并重排形成一种新型含氮平台化合物3-乙酰氨基-5-乙酰基呋喃（3A5AF），仅能通过化学催化合成。利用3A5AF为底物通过化学衍生已合成诸多高附加值含氮化学品。然而利用生物催化3A5AF合成其衍生物的报道仅有几例。Hao等^［136］首先以GlcNAc为底物化学合成了3A5AF，再利用羰基还原酶（ScCR）成功合成了手性3-乙酰氨基-5-（1-羟乙基）呋喃，具有优异的对映选择性。在此基础上，此团队进一步对ScCR进行了半理性改造，大大提高了其催化活性和稳定性，实现一锅法化学-酶两步催化合成了R-3-乙酰氨基-5-（1-羟乙基）呋喃，产率为53%，e.e.（对映体过量）>99%^［137］。陈可泉等^{［138-139］}以几丁质酶降解几丁质制备的GlcNAc经化学脱水合成了3A5AF，进一步利用筛选获得的转氨酶和脱乙酰酶通过不对称还原胺化和脱乙酰合成了126 mmol/L R-3-乙酰氨基-5-（1-氨基乙基）呋喃和47.58 mmol/L 2-乙酰基-4-氨基呋喃，转化率分别为84%（e.e.>99%）和79.3%。

4 结论

收起

几丁质是自然界中储量非常丰富的可再生资源，广泛存在于虾蟹壳和真菌细胞壁中，但由于缺乏高效的炼制手段，这些生物质多被作为废弃物处置，仅有少量作为饲料等底端产品进行高值化，导致巨大的资源浪费和环境污染。相比化学炼制，生物法具有过程条件温和、绿色及特异性好等优势，尤其是近年来随着合成生物学的快速崛起，进一步提升了生物法的实际应用前景。本综述总结了近些年来几丁质资源的生物降解和高值转化的进展，对几丁质资源的高效炼制具有重要意义。几丁质的生物炼制主要有两个方面：一方面是利用几丁质降解酶将几丁质水解为功能性寡糖（几丁寡糖和壳寡糖）和单糖（GlcNAc和氨基葡萄糖），因具有较好的生物活性，在医药、食品及农业等领域已经广泛应用；另一方面是基于几丁质的天然含氮元素的优势，利用微生物细胞或者酶将几丁质及其降解产物单糖或寡糖转化为N-乙酰神经氨酸、氨基酸及呋喃环类含氮化合物。因此，通过生物法将丰富的几丁质资源高效转化为高附加值产品具有保护环境和提高经济水平的双重意义，可为我国绿色可持续发展建设提供助力。

5 展望

收起

利用生物法对几丁质资源进行炼制，不但可实现废物利用，产生可观的经济效益，而且整个过程绿色环保，目前已经有诸多先进案例。然而，相较化学法，生物法仍存在效率低下、成本较高的瓶颈。因此，几丁质资源的高效生物炼制需要在以下几个方面不断改进：

首先，将几丁质降解为可溶性的寡糖和单糖是实现其高效生物炼制的关键，但目前缺乏高效的几丁质降解酶，对拥有结晶度高、分子量大结构特性的天然几丁质如虾蟹壳等的作用效果极差，因此以往的研究大量集于预处理方法开发，通过物理、化学等方法略降低几丁质结晶度，进而提高其酶解效率，但仍然存在一系列问题，例如物理法设备要求高、能耗高，化学法环境污染、条件苛刻等，均会导致成本较高，限制了其实际应用。目前报道中有少量新颖多功能几丁质酶，可以单酶高效降解天然几丁质如虾蟹壳等为几丁寡糖或GlcNAc，展现出较好的应用前景，但多种活性之间的不匹配，仍需要基于结构与功能关系的蛋白质工程改造提高其性能。因此，未来的研究核心之一是获得适于工业催化的、不需要对几丁质进行预处理的高效降解酶。例如通过特殊环境筛选或基于宏基因组学进行挖掘获得高性能天然酶，也可利用蛋白质工程对已有酶进行（半）理性改造和定向进化，结合高通量筛选方法，获得催化性能提升的优良突变体。

其次，目前报道中几丁质基含氮化合物多是利用化学法转化获得，主要是基于几丁质及其单体GlcNAc化学脱水合成的3-乙酰氨基-5-乙酰氨基呋喃（3A5AF）为平台，进一步通过还原胺化、还原、脱乙酰、异构化等化学反应合成一系列呋喃环含氮化学品。而相关化合物的生物催化合成案例较少，主要是受限于底物的非天然性，因此未来的研究第二个重要方向为基于天然的结构类似物催化酶对3A5AF转胺、还原等反应相关酶进行挖掘和工程改造，获得更多的含氮化学品合成相关酶元件和生物合成新路线。

此外，在几丁质合成含氮化学品的路线中，在不同步骤中生物法和化学法展现出各自的优缺点，未来的研究第三个重要方向是将生物法和化学法进行联用，充分发挥两种方法的各自优势和协同作用，建立高效转化几丁质资源到含氮化学品的生物-化学联合方法。例如利用酶法降解几丁质为GlcNAc，再采用化学法催化GlcNAc脱水环化形成3A5AF，进一步在酶的催化下合成呋喃环类含氮化合物。

随着合成生物学的迅速发展，以及人工智能的进一步辅助，相信在不久的将来，会实现低成本、绿色转化几丁质资源合成系列高附加值化学品。

基金

收起

国家重点研发计划(2021YFA0911400)

国家自然科学基金(22278220)

国家自然科学基金(U21B2097)

江苏省农业自主创新基金(CX(22)3070)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

1

Metal chlorides in ionic liquid solvents convert sugars to 5-hydroxymethylfurfural
ZHAO H B, HOLLADAY J E, BROWN H, et al. Metal chlorides in ionic liquid solvents convert sugars to 5-hydroxymethylfurfural[J]. Science, 2007, 316(5831): 1597-1600.

2

Extraction and characterization of chitin and chitosan from fishery waste by chemical method
KUMARI S, RATH P, HARI KUMAR A SRI, et al. Extraction and characterization of chitin and chitosan from fishery waste by chemical method[J]. Environmental Technology & Innovation, 2015, 3: 77-85.

3

CHITIN—a promising biomaterial for tissue engineering and stem cell technologies
WAN A C A, TAI B C U. CHITIN—a promising biomaterial for tissue engineering and stem cell technologies[J]. Biotechnology Advances, 2013, 31(8): 1776-1785.

4

Sustainability: don’t waste seafood waste
YAN N, CHEN X. Sustainability: don’t waste seafood waste[J]. Nature, 2015, 524(7564): 155-157.

5

王爱勤. 甲壳素化学[M]. 北京: 科学出版社, 2008.

WANG A Q. Chitin chemistry[M]. Beijing: Science Press, 2008.

6

Novel catalytic systems to convert chitin and lignin into valuable chemicals
CHEN X, YAN N. Novel catalytic systems to convert chitin and lignin into valuable chemicals[J]. Catalysis Surveys from Asia, 2014, 18(4): 164-176.

7

Green and sustainable manufacture of chemicals from biomass: state of the art
SHELDON R A. Green and sustainable manufacture of chemicals from biomass: state of the art[J]. Green Chemistry, 2014, 16(3): 950-963.

8

Recent trends in biological extraction of chitin from marine shell wastes: a review
KAUR S, DHILLON G S. Recent trends in biological extraction of chitin from marine shell wastes: a review[J]. Critical Reviews in Biotechnology, 2015, 35(1): 44-61.

9

Novel roles for GlcNAc in cell signaling
NASEEM S, PARRINO S M, BUENTEN D M, et al. Novel roles for GlcNAc in cell signaling[J]. Communicative & Integrative Biology, 2012, 5(2): 156-159.

10

Collagen-induced arthritis: severity and immune response attenuation using multivalent N-acetyl glucosamine
RICHTER J, CAPKOVÁ K, HŘÍBALOVÁ V, et al. Collagen-induced arthritis: severity and immune response attenuation using multivalent N-acetyl glucosamine[J]. Clinical and Experimental Immunology, 2014, 177(1): 121-133.

11

壳寡糖及衍生物抗肿瘤作用、免疫调节作用及其机制的研究
曹秀明. 壳寡糖及衍生物抗肿瘤作用、免疫调节作用及其机制的研究[D]. 青岛: 中国海洋大学, 2010.

CAO X M. Research on the anti-tumor and immunomodulatory effects and mechanisms of chitosan oligosaccharides and their derivatives [D]. Qingdao: Ocean University of China, 2010.

12

N-乙酰氨基葡萄糖治疗腹泻型肠易激综合征多中心临床研究
李兆申. N-乙酰氨基葡萄糖治疗腹泻型肠易激综合征多中心临床研究[J]. 中华消化杂志, 2009, 29(4): 267-270.

LI Z S. Treatment of diarrhea-predominant irritable bowel syndrome with N-acetyl-D-glucosamine: a randomized, double-blind, placebo-controlled multi-center study coorperative group[J]. Chinese Journal of Digestion, 2009, 29(4): 267-270.

13

几丁寡糖与壳寡糖制备和功能
张虎, 杜昱. 几丁寡糖与壳寡糖制备和功能[C]// 中国甲壳资源研究开发应用学术研讨会, 1997.

ZHANG H, DU Y. Preparation and function of chitosan oligosaccharides and chitosan oligosaccharides [C]// the academic symposium on the research, Development and Application of Chinese Crustacean Resources, 1997.

14

Preparation method of chitin oligosaccharide zinc borone magnesium fertilizer
SHAN J W, XUN J C. Preparation method of chitin oligosaccharide zinc borone magnesium fertilizer: CN1597634[P]. 2004-08-27.

15

Towards shell biorefinery: advances in chemical-catalytic conversion of chitin biomass to organonitrogen chemicals
DAI J H, LI F K, FU X. Towards shell biorefinery: advances in chemical-catalytic conversion of chitin biomass to organonitrogen chemicals[J]. ChemSusChem, 2020, 13(24): 6498-6508.

16

Expanding the boundary of biorefinery: organonitrogen chemicals from biomass
CHEN X, SONG S, LI H Y, et al. Expanding the boundary of biorefinery: organonitrogen chemicals from biomass[J]. Accounts of Chemical Research, 2021, 54(7): 1711-1722.

17

Catalytic conversion of chitin-based biomass to nitrogen-containing chemicals
JI X L, ZHAO Y F, LUI M Y, et al. Catalytic conversion of chitin-based biomass to nitrogen-containing chemicals[J]. iScience, 2024, 27(6): 109857.

18

Production of N-acetyl chitobiose from various chitin substrates using commercial enzymes
ILANKOVAN P, HEIN S, NG C H, et al. Production of N-acetyl chitobiose from various chitin substrates using commercial enzymes[J]. Carbohydrate Polymers, 2006, 63(2): 245-250.

19

Enzymatic hydrolysis of α-chitin
IL’INA A V, O Yu ZUEVA, LOPATIN S A, et al. Enzymatic hydrolysis of α-chitin[J]. Applied Biochemistry and Microbiology, 2004, 40(1): 35-38.

20

Chitin in protistan organisms: distribution, synthesis and deposition
MULISCH M. Chitin in protistan organisms: distribution, synthesis and deposition[J]. European Journal of Protistology, 1993, 29(1): 1-18.

21

从麻辣小龙虾虾壳中提取甲壳素的研究
伍军, 毛宏辉. 从麻辣小龙虾虾壳中提取甲壳素的研究[J]. 粮油加工, 2008(10): 128-130.

WU J, MAO H H. Study on extracting chitin from shell of spicy crayfish[J]. Cereals and Oils Processing, 2008(10): 128-130.

22

Facile production of chitin from shrimp shells using a deep eutectic solvent and acetic acid
ZHANG J, XU W R, ZHANG Y C. Facile production of chitin from shrimp shells using a deep eutectic solvent and acetic acid[J]. RSC Advances, 2022, 12(35): 22631-22638.

23

Facile production of chitin from crab shells using ionic liquid and citric acid
SETOGUCHI T, KATO T, YAMAMOTO K, et al. Facile production of chitin from crab shells using ionic liquid and citric acid[J]. International Journal of Biological Macromolecules, 2012, 50(3): 861-864.

24

One-pot production of chitin with high purity from lobster shells using choline chloride-malonic acid deep eutectic solvent
ZHU P, GU Z J, HONG S, et al. One-pot production of chitin with high purity from lobster shells using choline chloride-malonic acid deep eutectic solvent[J]. Carbohydrate Polymers, 2017, 177: 217-223.

25

Efficient decomposition of shrimp shell waste using Bacillus cereus and Exiguobacterium acetylicum
SOROKULOVA I, KRUMNOW A, GLOBA L, et al. Efficient decomposition of shrimp shell waste using Bacillus cereus and Exiguobacterium acetylicum [J]. Journal of Industrial Microbiology & Biotechnology, 2009, 36(8): 1123-1126.

26

以嗜酸乳杆菌SWO1发酵虾头虾壳提取蛋白质和甲壳素的工艺研究
李磊. 以嗜酸乳杆菌SWO1发酵虾头虾壳提取蛋白质和甲壳素的工艺研究[D]. 广州: 华南农业大学, 2011.

LI L. Study on the process of extracting protein and chitin from shrimp head shells using Lactobacillus acidophilus SWO1 fermentation[D]. Guangzhou: South China Agricultural University, 2011.

27

植物乳杆菌发酵虾头、虾壳回收蛋白质和甲壳素的研究
刘斯雅, 林瑞君, 庄泽娟, 等. 植物乳杆菌发酵虾头、虾壳回收蛋白质和甲壳素的研究[J]. 现代食品科技, 2011, 27(4): 408-411, 383.

LIU S Y, LIN R J, ZHUANG Z J, et al. Recovery of protein and chitin from shrimp waste by lactic acid fermentation with l.plantarum[J]. Modern Food Science and Technology, 2011, 27(4): 408-411, 383.

28

Recovery of chitin and chitosan from shrimp waste by chemical and microbial methods
KHANAFARI A, MARANDI R, SANATEI S. Recovery of chitin and chitosan from shrimp waste by chemical and microbial methods[J]. Iranian Journal of Enviromental Health Science & Engineering, 2008, 5(1): 19-24.

29

Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources
JANG M K, KONG B G, JEONG Y I, et al. Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources[J]. Journal of Polymer Science Part A: Polymer Chemistry, 2004, 42(14): 3423-3432.

30

Influence of alkali-freezing treatment on the solid state structure of chitin
FENG F, LIU Y, HU K A. Influence of alkali-freezing treatment on the solid state structure of chitin[J]. Carbohydrate Research, 2004, 339(13): 2321-2324.

31

Elucidating the internal structure and dynamics of α-chitin by 2DPASS-MAS-NMR and spin-lattice relaxation measurements
GHOSH M, SADHUKHAN S, DEY K K. Elucidating the internal structure and dynamics of α-chitin by 2DPASS-MAS-NMR and spin-lattice relaxation measurements[J]. Solid State Nuclear Magnetic Resonance, 2019, 97: 7-16.

32

Histochemical evidence of β-chitin in parapodial glandular organs and tubes of Spiophanes (Annelida, Sedentaria: Spionidae), and first studies on selected Annelida
GUGGOLZ T, HENNE S, POLITI Y, et al. Histochemical evidence of β-chitin in parapodial glandular organs and tubes of Spiophanes (Annelida, Sedentaria: Spionidae), and first studies on selected Annelida[J]. Journal of Morphology, 2015, 276(12): 1433-1447.

33

Refinement of the structure of beta-chitin
GARDNER K H, BLACKWELL J. Refinement of the structure of beta-chitin[J]. Biopolymers, 1975, 14(8): 1581-1595.

34

A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation
SUBRAMANI A K, RAVAL R, SUNDARESHAN S, et al. A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation[J]. Preparative Biochemistry & Biotechnology, 2022, 52(10): 1160-1172.

35

Biotechnological eminence of chitinases: a focus on thermophilic enzyme sources, production strategies and prominent applications
AKRAM F, AKRAM R, UL HAQ I, et al. Biotechnological eminence of chitinases: a focus on thermophilic enzyme sources, production strategies and prominent applications[J]. Protein and Peptide Letters, 2021, 28(9): 1009-1022.

36

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus
JUPATANAKUL N, PENGON J, SELISANA S M G, et al. Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus [J]. Acta Tropica, 2020, 212: 105686.

37

Purification and characterization of a 34-kDa chitobiosidase from Aeromonas sp. GJ-18
JEONG H C, JU W T, JO K H, et al. Purification and characterization of a 34-kDa chitobiosidase from Aeromonas sp. GJ-18[J]. Journal of the Korean Society for Applied Biological Chemistry, 2012, 55(1): 7-12.

38

The draft genome sequence and analysis of an efficiently chitinolytic bacterium Chitinibacter sp. strain GC72
ZHANG A L, MO X F, WEI G G, et al. The draft genome sequence and analysis of an efficiently chitinolytic bacterium Chitinibacter sp. strain GC72[J]. Current Microbiology, 2020, 77(12): 3903-3908.

39

Identification of an endo-chitinase from Corallococcus sp. EGB and evaluation of its antifungal properties
LI Z K, XIA C Y, WANG Y X, et al. Identification of an endo-chitinase from Corallococcus sp. EGB and evaluation of its antifungal properties[J]. International Journal of Biological Macromolecules, 2019, 132: 1235-1243.

40

Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant
HAO Z K, LI J S, WANG D H, et al. Efficient production of GlcNAc in an aqueous-organic system with a Chitinolyticbacter meiyuanensis SYBC-H1 mutant[J]. Biotechnology Letters, 2022, 44(4): 623-633.

41

Production of chitinase by Fusarium species
NUERO O M. Production of chitinase by Fusarium species[J]. Current Microbiology, 1995, 30(5): 287-289.

42

Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation
BINOD P, PUSZTAHELYI T, NAGY V, et al. Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation[J]. Enzyme and Microbial Technology, 2005, 36(7): 880-887.

43

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications
KHAN F I, BISETTY K, SINGH S, et al. Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications[J]. Extremophiles, 2015, 19(6): 1055-1066.

44

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum
PRABAVATHY V R, MATHIVANAN N, SAGADEVAN E, et al. Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum [J]. Bioresource Technology, 2006, 97(18): 2330-2334.

45

Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens
GAO F, ZHANG B S, ZHAO J H, et al. Deacetylation of chitin oligomers increases virulence in soil-borne fungal pathogens[J]. Nature Plants, 2019, 5(11): 1167-1176.

46

2-Naphthol impregnation prior to steam explosion promotes LPMO-assisted enzymatic saccharification of spruce and yields high-purity lignin
HANSEN L D, ØSTENSEN M, ARSTAD B, et al. 2-Naphthol impregnation prior to steam explosion promotes LPMO-assisted enzymatic saccharification of spruce and yields high-purity lignin[J]. ACS Sustainable Chemistry & Engineering, 2022, 10(16): 5233-5242.

47

LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus
LIU F, SELIN C, ZOU Z W, et al. LmCBP1, a secreted chitin-binding protein, is required for the pathogenicity of Leptosphaeria maculans on Brassica napus [J]. Fungal Genetics and Biology, 2020, 136: 103320.

48

Erratum: molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae
THOMPSON C E. Erratum: molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae [J]. Genetics and Molecular Biology, 2019, 42(1): 151.

49

Updating the sequence-based classification of glycosyl hydrolases
HENRISSAT B, BAIROCH A. Updating the sequence-based classification of glycosyl hydrolases[J]. Biochemical Journal, 1996, 316(Pt 2): 695-696.

50

Cloning and characterization of a novel GH75 family chitosanase from Penicillium oxalicum M2
CAO S N, GAO P, XIA W S, et al. Cloning and characterization of a novel GH75 family chitosanase from Penicillium oxalicum M2[J]. Process Biochemistry, 2022, 120: 41-52.

51

微生物几丁质酶研究进展
王治伟, 刘志敏. 微生物几丁质酶研究进展[J]. 生物技术通讯, 2006, 17(3): 439-442.

WANG Z W, LIU Z M. Advance in study and application on chitinase produced by microbes[J]. Letters in Biotechnology, 2006, 17(3): 439-442.

52

Characterization of a new multifunctional GH20 β-N-acetylglucosaminidase from Chitinibacter sp. GC72 and its application in converting chitin into N-acetyl glucosamine
CHEN Y, ZHOU N, CHEN X M, et al. Characterization of a new multifunctional GH20 β-N-acetylglucosaminidase from Chitinibacter sp. GC72 and its application in converting chitin into N-acetyl glucosamine[J]. Frontiers in Microbiology, 2022, 13: 874908.

53

A novel thermostable chitinolytic machinery of Streptomyces sp. F-3 consisting of chitinases with different action modes
SUN X M, LI Y J, TIAN Z N, et al. A novel thermostable chitinolytic machinery of Streptomyces sp. F-3 consisting of chitinases with different action modes[J]. Biotechnology for Biofuels, 2019, 12: 136.

54

Morphological and structural characterization of chitin as a substrate for the screening, production, and molecular characterization of chitinase by Bacillus velezensis
DAHIYA D, PILLI A, CHIRRA P R R, et al. Morphological and structural characterization of chitin as a substrate for the screening, production, and molecular characterization of chitinase by Bacillus velezensis [J]. Environmental Science and Pollution Research, 2022, 29(57): 86550-86561.

55

From natural microbe screening to sustained chitinase activity in exogenous hosts
CHAULAGAIN D, SHAMABADI N S, LESLIE S A, et al. From natural microbe screening to sustained chitinase activity in exogenous hosts[J]. ACS Synthetic Biology, 2024, 13(4): 1165-1176.

56

Identification of chitinolytic enzymes in Chitinolyticbacter meiyuanensis and mechanism of efficiently hydrolyzing chitin to N-acetyl glucosamine
ZHANG A L, MO X F, ZHOU N, et al. Identification of chitinolytic enzymes in Chitinolyticbacter meiyuanensis and mechanism of efficiently hydrolyzing chitin to N-acetyl glucosamine[J]. Frontiers in Microbiology, 2020, 11: 572053.

57

Chitinolyticbacter meiyuanensis的筛选鉴定及其发酵产几丁质酶研究
郝之奎. Chitinolyticbacter meiyuanensis的筛选鉴定及其发酵产几丁质酶研究[D]. 无锡: 江南大学, 2011.

HAO Z K. Screening and identification of Chitinolyticbacter meiyuanensis and its fermentation for chitinase production[D]. Wuxi: Jiangnan University, 2011.

58

NetSyn: genomic context exploration of protein families
STAM M, LANGLOIS J, CHEVALIER C, et al. NetSyn: genomic context exploration of protein families[EB/OL]. bioRxiv, 2023, 2023.02.15.528638. (2023-02-15)[2024-03-01] . https://doi.org/10.1101/2023.02.15.528638

59

Knock down of chitosanase expression in phytopathogenic fungus Fusarium solani and its effect on pathogenicity
LIU H W, ZHANG B, LI C S, et al. Knock down of chitosanase expression in phytopathogenic fungus Fusarium solani and its effect on pathogenicity[J]. Current Genetics, 2010, 56(3): 275-281.

60

Chitinolytic functions in Actinobacteria: ecology, enzymes, and evolution
LACOMBE-HARVEY M È, BRZEZINSKI R, BEAULIEU C. Chitinolytic functions in Actinobacteria: ecology, enzymes, and evolution[J]. Applied Microbiology and Biotechnology, 2018, 102(17): 7219-7230.

61

Biochemical characterization of a GH19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol
LV C Y, GU T Y, MA R, et al. Biochemical characterization of a GH19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol[J]. International Journal of Biological Macromolecules, 2021, 167: 193-201.

62

枯草芽孢杆菌B-908几丁质酶基因的转化及表达
程爱丽, 唐文华, 王益民. 枯草芽孢杆菌B-908几丁质酶基因的转化及表达[J]. 植物病理学报, 1996, 26(3): 204.

CHENG A L, TANG W H, WANG Y M. The transformation and expression of chitinase gene from Bacillus subtilis B-908[J]. Acta Phytopathologica Sinica, 1996, 26(3): 204.

63

Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides
VAIKUNTAPU P R, MALLAKUNTLA M K, DAS S N, et al. Applicability of endochitinase of Flavobacterium johnsoniae with transglycosylation activity in generating long-chain chitooligosaccharides[J]. International Journal of Biological Macromolecules, 2018, 117: 62-71.

64

Discovering and designing a chimeric hyperthermophilic chitinase for crystalline chitin degradation
SUN B, ZHAO X C, XU B R, et al. Discovering and designing a chimeric hyperthermophilic chitinase for crystalline chitin degradation[J]. ACS Sustainable Chemistry & Engineering, 2023, 11(12): 4690-4698.

65

High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris
CHEN X M, ZHAI C, KANG L X, et al. High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris [J]. Biotechnology Letters, 2012, 34(4): 689-694.

66

Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6
WANG X H, CHI N Y, BAI F W, et al. Characterization of a cold-adapted and salt-tolerant exo-chitinase (ChiC) from Pseudoalteromonas sp. DL-6[J]. Extremophiles, 2016, 20(2): 167-176.

67

N-acetyl-D-glucosamine production by a chitinase of marine fungal origin: a case study of potential industrial significance for valorization of waste chitins
DAS S, DEY P, ROY D, et al. N-acetyl-D-glucosamine production by a chitinase of marine fungal origin: a case study of potential industrial significance for valorization of waste chitins[J]. Applied Biochemistry and Biotechnology, 2019, 187(1): 407-423.

68

Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii
YANG S Q, FU X, YAN Q J, et al. Cloning, expression, purification and application of a novel chitinase from a thermophilic marine bacterium Paenibacillus barengoltzii [J]. Food Chemistry, 2016, 192: 1041-1048.

69

Biochemical characterization of a novel hyperthermophilic chitinase from a deep-sea Thermotogae bacterium
XU Y, OUYANG B, DENG L Y, et al. Biochemical characterization of a novel hyperthermophilic chitinase from a deep-sea Thermotogae bacterium[J]. Process Biochemistry, 2024, 143: 60-72.

70

微生物几丁质酶研究进展及应用
王琳, 陈雅如, 程湄婕, 等. 微生物几丁质酶研究进展及应用[J]. 中国生物工程杂志, 2022, 42(12): 101-110.

WANG L, CHEN Y R, CHENG M J, et al. Research advances in microbial chitinase and its applications[J]. China Biotechnology, 2022, 42(12): 101-110.

71

Acid hydrolysis of chitin and chitosan
NOVIKOV V Y. Acid hydrolysis of chitin and chitosan[J]. Russian Journal of Applied Chemistry, 2004, 77(3): 484-487.

72

Accelerating enzymatic hydrolysis of chitin by microwave pretreatment
ROY I, MONDAL K, GUPTA M N. Accelerating enzymatic hydrolysis of chitin by microwave pretreatment[J]. Biotechnology Progress, 2003, 19(6): 1648-1653.

73

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation
XING R E, LIU S, YU H H, et al. Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation[J]. Carbohydrate Research, 2005, 340(13): 2150-2153.

74

Engineering a carbohydrate binding module to enhance chitinase catalytic efficiency on insoluble chitinous substrate
SU H P, GAO L, SUN J A, et al. Engineering a carbohydrate binding module to enhance chitinase catalytic efficiency on insoluble chitinous substrate[J]. Food Chemistry, 2021, 355: 129462.

75

人工构建几丁质小体多酶复合体scaford-chiC-chiA-sg的方法及应用
陈可泉, 周宁, 张阿磊, 等. 人工构建几丁质小体多酶复合体scaford-chiC-chiA-sg的方法及应用: CN112522246A[P]. 2021-03-19.

CHEN K Q, ZHOU N, ZHANG A L, et al. Artificial construction of scaford-chiC-chiA-sg, a multi-enzyme complex of chitinosomes, and its application: CN112522246A[P]. 2021-03-19.

76

Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion
DENG J J, SHI D, MAO H H, et al. Heterologous expression and characterization of an antifungal chitinase (Chit46) from Trichoderma harzianum GIM 3.442 and its application in colloidal chitin conversion[J]. International Journal of Biological Macromolecules, 2019, 134: 113-121.

77

Structure-function insights into the fungal endo-chitinase Chit33 depict its mechanism on chitinous material
JIMÉNEZ-ORTEGA E, KIDIBULE P E, FERNÁNDEZ-LOBATO M, et al. Structure-function insights into the fungal endo-chitinase Chit33 depict its mechanism on chitinous material[J]. International Journal of Molecular Sciences, 2022, 23(14): 7599.

78

Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-D-glucosamine production
ZHANG A L, HE Y M, WEI G G, et al. Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-D-glucosamine production[J]. Biotechnology for Biofuels, 2018, 11: 179.

79

Overexpression of the chimeric chitinase (ChBD) gene in Lycopersicon esculentum L. enhanced resistance to Sclerotinia sclerotiorum
GOUGERDCHI V, DORANI E, VALIZADEH M, et al. Overexpression of the chimeric chitinase (ChBD) gene in Lycopersicon esculentum L. enhanced resistance to Sclerotinia sclerotiorum [J]. Plant Cell, Tissue and Organ Culture, 2022, 151(1): 165-175.

80

Increased antifungal activity of Chit42 from Trichoderma atroviride by addition of a chitin binding domain
ATAEI A, ZAMANI M, MOTALLEBI M, et al. Increased antifungal activity of Chit42 from Trichoderma atroviride by addition of a chitin binding domain[J]. Tropical Plant Pathology, 2016, 41(6): 350-356.

81

Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β- endoglucanase
YAN J J, LIU W D, LI Y J, et al. Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β- endoglucanase[J]. Biochemical and Biophysical Research Communications, 2016, 475(1): 8-12.

82

Genome-wide identification and analysis of chitinase GH18 gene family in Mycogone perniciosa
YANG Y, SOSSAH F L, LI Z, et al. Genome-wide identification and analysis of chitinase GH18 gene family in Mycogone perniciosa [J]. Frontiers in Microbiology, 2021, 11: 596719.

83

An optimized cocktail of chitinolytic enzymes to produce N,N'-diacetylchitobiose and N-acetyl-D-glucosamine from defatted krill by-products
CHU F M, WANG D, LIU T, et al. An optimized cocktail of chitinolytic enzymes to produce N,N'-diacetylchitobiose and N-acetyl-D-glucosamine from defatted krill by-products[J]. International Journal of Biological Macromolecules, 2019, 133: 1029-1034.

84

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin
NAKAMURA A, OKAZAKI K I, FURUTA T, et al. Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin[J]. Nature Communications, 2018, 9(1): 3814.

85

Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste
SONGSIRIRITTHIGUL C, LAPBOONRUENG S, PECHSRICHUANG P, et al. Expression and characterization of Bacillus licheniformis chitinase (ChiA), suitable for bioconversion of chitin waste[J]. Bioresource Technology, 2010, 101(11): 4096-4103.

86

Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H
MENGHIU G, OSTAFE V, PRODANOVIC R, et al. Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H[J]. Protein Expression and Purification, 2019, 154: 25-32.

87

甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖
潘梦妍, 徐显皓, 刘延峰, 等. 甲壳素酶Chisb的定向进化及生物转化合成几丁寡糖[J]. 生物工程学报, 2019, 35(9): 1787-1796.

PAN M Y, XU X H, LIU Y F, et al. Directed evolution of chitinase Chisb and biosynthesis of chitooligosaccharides[J]. Chinese Journal of Biotechnology, 2019, 35(9): 1787-1796.

88

Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design
XU P, NI Z F, ZONG M H, et al. Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design[J]. International Journal of Biological Macromolecules, 2020, 150: 9-15.

89

Stabilization of a chitinase from Serratia marcescens by Gly→Ala and Xxx→Pro mutations
GÅSEIDNES S, SYNSTAD B, JIA X H, et al. Stabilization of a chitinase from Serratia marcescens by Gly→Ala and Xxx→Pro mutations[J]. Protein Engineering, 2003, 16(11): 841-846.

90

Engineering the relatively conserved residues in active site architecture of thermophilic chitinase SsChi18A enhanced catalytic activity
ZHAO S, LIU M Y, SUN X M, et al. Engineering the relatively conserved residues in active site architecture of thermophilic chitinase SsChi18A enhanced catalytic activity[J]. Biomacromolecules, 2024, 25(1): 238-247.

91

Carbohydrate-binding modules of ChiB and ChiC promote the chitinolytic system of Serratia marcescens BWL1001
LIU J W, XU Q, WU Y, et al. Carbohydrate-binding modules of ChiB and ChiC promote the chitinolytic system of Serratia marcescens BWL1001[J]. Enzyme and Microbial Technology, 2023, 162: 110118.

92

Conversion of shrimp head waste for production of a thermotolerant, detergent-stable, alkaline protease by Paenibacillus sp
DOAN C T, TRAN T N, WEN I H, et al. Conversion of shrimp head waste for production of a thermotolerant, detergent-stable, alkaline protease by Paenibacillus sp[J]. Catalysts, 2019, 9(10): 798.

93

解淀粉芽孢杆菌壳聚糖酶毕赤酵母高效表达
罗洒, 秦臻, 陈启明, 等. 解淀粉芽孢杆菌壳聚糖酶毕赤酵母高效表达[C]//中国食品科学技术学会第十五届年会论文集. 青岛, 2018: 436.

LUO S, QIN Z, CHEN Q M, et al. Efficient expression of chitosan enzyme from Bacillus amyloliquefaciens in Pichia pastoris [C]// Proceedings of the 15th Annual Conference of the Chinese Society for Food Science and Technology. Qingdao, 2018: 436.

94

Biocatalysis of heterogenously-expressed chitosanase for the preparation of desirable chitosan oligosaccharides applied against phytopathogenic fungi
ZHOU J L, LIU X B, YUAN F, et al. Biocatalysis of heterogenously-expressed chitosanase for the preparation of desirable chitosan oligosaccharides applied against phytopathogenic fungi[J]. ACS Sustainable Chemistry & Engineering, 2020, 8(12): 4781-4791.

95

壳寡糖的制备与应用研究进展
李茜茜, 杨云洁, 管世敏, 等. 壳寡糖的制备与应用研究进展[J]. 粮食与油脂, 2023, 36(9): 27-31.

LI Q Q, YANG Y J, GUAN S M, et al. Research progress on the preparation and application of chitosan oligosaccharides[J]. Cereals & Oils, 2023, 36(9): 27-31.

96

Chitosanase-immobilized magnetite-agar gel particles as a highly stable and reusable biocatalyst for enhanced production of physiologically active chitosan oligosaccharides
KUROIWA T, NAKAGAWA Y, TAKAYANAGI R, et al. Chitosanase-immobilized magnetite-agar gel particles as a highly stable and reusable biocatalyst for enhanced production of physiologically active chitosan oligosaccharides[J]. Enzyme and Microbial Technology, 2024, 178: 110443.

97

Characterization of a chitinase from Salinivibrio sp. BAO-1801 as an antifungal activity and a biocatalyst for producing chitobiose
LE B, YANG S H. Characterization of a chitinase from Salinivibrio sp. BAO-1801 as an antifungal activity and a biocatalyst for producing chitobiose[J]. Journal of Basic Microbiology, 2018, 58(10): 848-856.

98

Biochemical properties of a cold-active chitinase from marine Trichoderma gamsii R1 and its application to preparation of chitin oligosaccharides
WANG J R, ZHU M J, WANG P, et al. Biochemical properties of a cold-active chitinase from marine Trichoderma gamsii R1 and its application to preparation of chitin oligosaccharides[J]. Marine Drugs, 2023, 21(6): 332.

99

Expression and characterization of a chitinase from Serratia marcescens
LI J C, ZHENG J M, LIANG Y H, et al. Expression and characterization of a chitinase from Serratia marcescens [J]. Protein Expression and Purification, 2020, 171: 105613.

100

β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin
KROLICKA M, HINZ S W A, KOETSIER M J, et al. β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin[J]. Applied Microbiology and Biotechnology, 2018, 102(17): 7441-7454.

101

The discovery, enzymatic characterization and functional analysis of a newly isolated chitinase from marine-derived fungus Aspergillus fumigatus df347
WU Y L, WANG S, YANG D F, et al. The discovery, enzymatic characterization and functional analysis of a newly isolated chitinase from marine-derived fungus Aspergillus fumigatus df347[J]. Marine Drugs, 2022, 20(8): 520.

102

Purification and characterization of a chitinase from Aeromonas media CZW001 as a biocatalyst for producing chitinpentaose and chitinhexaose
DING Z W, LI T, CHEN M, et al. Purification and characterization of a chitinase from Aeromonas media CZW001 as a biocatalyst for producing chitinpentaose and chitinhexaose[J]. Biotechnology and Applied Biochemistry, 2023, 70(1): 281-289.

103

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production
SUBRAMANI A K, RAMACHANDRA R, THOTE S, et al. Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production[J]. International Journal of Biological Macromolecules, 2024, 264(Pt 2): 130499.

104

One-step processing of shrimp shell waste with a chitinase fused to a carbohydrate-binding module
DENG J J, ZHANG M S, LI Z W, et al. One-step processing of shrimp shell waste with a chitinase fused to a carbohydrate-binding module[J]. Green Chemistry, 2020, 22(20): 6862-6873.

105

维氏气单胞菌来源几丁质酶的克隆表达及应用
鲁梦唯, 陈晟, 吴敬. 维氏气单胞菌来源几丁质酶的克隆表达及应用[J]. 食品与生物技术学报, 2022, 41(4): 55-63.

LU M W, CHEN S, WU J. Cloning, expression and application of chitinase from Aeromonas veronii [J]. Journal of Food Science and Biotechnology, 2022, 41(4): 55-63.

106

A transglycosylating chitinase from Chitiniphilus shinanonensis (CsChiL) hydrolyzes chitin in a processive manner
BHUVANACHANDRA B, PODILE A R. A transglycosylating chitinase from Chitiniphilus shinanonensis (CsChiL) hydrolyzes chitin in a processive manner[J]. International Journal of Biological Macromolecules, 2020, 145: 1-10.

107

Glycosidase-catalysed oligosaccharide synthesis: preparation of the N-acetylchitooligosaccharidespenta-N-acetylchitopentaose and hexa-N-acetylchitohexaose using the β-N-acetylhexosaminidase of Aspergillus oryzae
SINGH S, GALLAGHER R, DERRICK P J, et al. Glycosidase-catalysed oligosaccharide synthesis: preparation of the N-acetylchitooligosaccharidespenta-N-acetylchitopentaose and hexa-N-acetylchitohexaose using the β-N-acetylhexosaminidase of Aspergillus oryzae [J]. Tetrahedron: Asymmetry, 1995, 6(11): 2803-2810.

108

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities
ZHANG A L, MO X F, ZHOU N, et al. A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities[J]. Biotechnology for Biofuels, 2020, 13: 115.

109

木瓜蛋白酶法制备抗氧化活性壳寡糖的工艺优化
黄晓月, 毕思远, 区家豪, 等. 木瓜蛋白酶法制备抗氧化活性壳寡糖的工艺优化[J]. 生物学杂志, 2022, 39(1): 104-109.

HUANG X Y, BI S Y, OU J H, et al. Process optimization of preparation of antioxidant chitooligosaccharides by papain[J]. Journal of Biology, 2022, 39(1): 104-109.

110

聚合度6-8壳寡糖的制备关键技术研究
董惠忠. 聚合度6-8壳寡糖的制备关键技术研究[D]. 上海: 华东理工大学, 2014.

DONG H Z. Key technology research on the preparation of chitosan oligosaccharides with a polymerization degree of 6-8[D]. Shanghai: East China University of Science and Technology, 2014.

111

An efficient enzymatic production of N-acetyl-D-glucosamine from crude chitin powders
ZHANG A L, GAO C, WANG J, et al. An efficient enzymatic production of N-acetyl-D-glucosamine from crude chitin powders[J]. Green Chemistry, 2016, 18(7): 2147-2154.

112

Enzymatic hydrolysis of chitin pretreated by bacterial fermentation to obtain pure N-acetyl-D-glucosamine
ZHANG A L, WEI G G, MO X F, et al. Enzymatic hydrolysis of chitin pretreated by bacterial fermentation to obtain pure N-acetyl-D-glucosamine[J]. Green Chemistry, 2018, 20(10): 2320-2327.

113

The effect of ultrasonication on enzymatic hydrolysis of chitin to N-acetyl glucosamine via sequential and simultaneous strategies
WANG Y Y, ZHANG A L, MO X F, et al. The effect of ultrasonication on enzymatic hydrolysis of chitin to N-acetyl glucosamine via sequential and simultaneous strategies[J]. Process Biochemistry, 2020, 99: 265-269.

114

Enzymatic production of N-acetyl-D-glucosamine from crayfish shell wastes pretreated via high pressure homogenization
WEI G G, ZHANG A L, CHEN K Q, et al. Enzymatic production of N-acetyl-D-glucosamine from crayfish shell wastes pretreated via high pressure homogenization[J]. Carbohydrate Polymers, 2017, 171: 236-241.

115

Effect of organic solvents treatment on structure of chitin and its enzymatic hydrolysis
ZHOU N, YANG P F, CHEN J, et al. Effect of organic solvents treatment on structure of chitin and its enzymatic hydrolysis[J]. Polymer Degradation and Stability, 2022, 198: 109654.

116

Efficient enzymatic hydrolysis of chitin into N-acetyl glucosamine using alkali as a recyclable pretreatment reagent
ZHANG A L, WANG C Y, CHEN J, et al. Efficient enzymatic hydrolysis of chitin into N-acetyl glucosamine using alkali as a recyclable pretreatment reagent[J]. Green Chemistry, 2021, 23(8): 3081-3089.

117

Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates
CARDOZO F A, GONZALEZ J M, FEITOSA V A, et al. Bioconversion of α-chitin into N-acetyl-glucosamine using chitinases produced by marine-derived Aeromonas caviae isolates[J]. World Journal of Microbiology & Biotechnology, 2017, 33(11): 201.

118

Enzymatic processing of chitinaceous wastes for N-acetyl-D-glucosamine production: an example of green and efficient environmental management
DAS S, SEN R, ROY D. Enzymatic processing of chitinaceous wastes for N-acetyl-D-glucosamine production: an example of green and efficient environmental management[J]. Environmental Engineering and Management Journal, 2012, 11(10): 1849-1855.

119

Efficient enzymatic hydrolysis of ionic liquid pretreated chitin and its dissolution mechanism
LI J, HUANG W C, GAO L, et al. Efficient enzymatic hydrolysis of ionic liquid pretreated chitin and its dissolution mechanism[J]. Carbohydrate Polymers, 2019, 211: 329-335.

120

Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin
NGUYEN-THI N, DOUCET N. Combining chitinase C and N-acetylhexosaminidase from Streptomyces coelicolor A3(2) provides an efficient way to synthesize N-acetylglucosamine from crystalline chitin[J]. Journal of Biotechnology, 2016, 220: 25-32.

121

Biochemical characterization of two β-N-acetylglucosaminidases from Streptomyces violascens for efficient production of N-acetyl-D-glucosamine
LI J, GAO K P, SECUNDO F, et al. Biochemical characterization of two β-N-acetylglucosaminidases from Streptomyces violascens for efficient production of N-acetyl-D-glucosamine[J]. Food Chemistry, 2021, 364: 130393.

122

Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc
SONG W, ZHANG N, YANG M, et al. Multiple strategies to improve the yield of chitinase a from Bacillus licheniformis in Pichia pastoris to obtain plant growth enhancer and GlcNAc[J]. Microbial Cell Factories, 2020, 19(1): 181.

123

Bacterial surface-assembled chitinosome for dismantling chitin into N-acetyl glucosamine
DU C, ZHOU Y L, LIU L, et al. Bacterial surface-assembled chitinosome for dismantling chitin into N-acetyl glucosamine[J]. ACS Sustainable Chemistry & Engineering, 2023, 11(30): 11239-11247.

124

A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T
OKAZAKI S, KOMATSU A, NAKANO M, et al. A novel endo-type chitinase possessing chitobiase activity derived from the chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T[J]. Bioscience, Biotechnology, and Biochemistry, 2023, 87(12): 1543-1550.

125

A Bacillus pumilus originated β-N-acetylglucosaminidase for chitin combinatory hydrolysis and exploration of its thermostable mechanism
DU C, JIANG S, JIANG S J, et al. A Bacillus pumilus originated β-N-acetylglucosaminidase for chitin combinatory hydrolysis and exploration of its thermostable mechanism[J]. International Journal of Biological Macromolecules, 2019, 132: 1282-1289.

126

Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-β-D-glucosamine from powdery chitin
HAN S S, XUE Y B, YAN Q J, et al. Development of a two-enzyme system in Aspergillus niger for efficient production of N-acetyl-β-D-glucosamine from powdery chitin[J]. Bioresource Technology, 2024, 393: 130024.

127

An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine
FU X, YAN Q J, YANG S Q, et al. An acidic, thermostable exochitinase with β-N-acetylglucosaminidase activity from Paenibacillus barengoltzii converting chitin to N-acetyl glucosamine[J]. Biotechnology for Biofuels, 2014, 7(1): 174.

128

Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi
SURESH P V, ANIL KUMAR P K. Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi[J]. Biodegradation, 2012, 23(4): 597-607.

129

Production of N-acetyl-D-glucosamine from mycelial waste by a combination of bacterial chitinases and an insect N-acetyl-D-glucosaminidase
ZHU W X, WANG D, LIU T, et al. Production of N-acetyl-D-glucosamine from mycelial waste by a combination of bacterial chitinases and an insect N-acetyl-D-glucosaminidase[J]. Journal of Agricultural and Food Chemistry, 2016, 64(35): 6738-6744.

130

Characterization of extracellular chitinase from Chitinibacter sp. GC72 and its application in GlcNAc production from crayfish shell enzymatic degradation
GAO C, ZHANG A L, CHEN K Q, et al. Characterization of extracellular chitinase from Chitinibacter sp. GC72 and its application in GlcNAc production from crayfish shell enzymatic degradation[J]. Biochemical Engineering Journal, 2015, 97: 59-64.

131

一种几丁质脱乙酰基酶突变体及其编码基因与应用
吕永梅, 章晓洋, 高文博, 等. 一种几丁质脱乙酰基酶突变体及其编码基因与应用: CN116144636A[P]. 2023-05-23.

LV Y M, ZHANG X Y, GAO W B, et al. A chitin deacetylase mutant and its coding gene and application: CN116144636A[P]. 2023-05-23.

132

Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species
INOKUMA K, TAKANO M, HOSHINO K. Direct ethanol production from N-acetylglucosamine and chitin substrates by Mucor species[J]. Biochemical Engineering Journal, 2013, 72: 24-32.

133

An excellent anaerobic respiration mode for chitin degradation by Shewanella oneidensis MR-1 in microbial fuel cells
LI S W, ZENG R J, SHENG G P. An excellent anaerobic respiration mode for chitin degradation by Shewanella oneidensis MR-1 in microbial fuel cells[J]. Biochemical Engineering Journal, 2017, 118: 20-24.

134

One-pot biosynthesis of N-acetylneuraminic acid from chitin via combination of chitin-degrading enzymes, N-acetylglucosamine-2-epimerase, and N-neuraminic acid aldolase
LIU Q Z, WEI G G, YANG P F, et al. One-pot biosynthesis of N-acetylneuraminic acid from chitin via combination of chitin-degrading enzymes, N-acetylglucosamine-2-epimerase, and N-neuraminic acid aldolase[J]. Frontiers in Microbiology, 2023, 14: 1156924.

135

Upcycling chitin-containing waste into organonitrogen chemicals via an integrated process
MA X Q, GÖZAYDIN G, YANG H Y, et al. Upcycling chitin-containing waste into organonitrogen chemicals via an integrated process[J]. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(14): 7719-7728.

136

Chemoenzymatic access to enantiopure N-containing furfuryl alcohol from chitin-derived N-acetyl-D-glucosamine
HAO Y C, ZONG M H, WANG Z L, et al. Chemoenzymatic access to enantiopure N-containing furfuryl alcohol from chitin-derived N-acetyl-D-glucosamine[J]. Bioresources and Bioprocessing, 2021, 8(1): 80.

137

Engineering carbonyl reductase for one-pot chemobiocatalytic enantioselective synthesis of a value-added N-containing chiral alcohol from N-acetyl-D-glucosamine
HAO Y C, ZONG M H, CHEN Q, et al. Engineering carbonyl reductase for one-pot chemobiocatalytic enantioselective synthesis of a value-added N-containing chiral alcohol from N-acetyl-D-glucosamine[J]. Green Chemistry, 2023, 25(13): 5051-5058.

138

陈可泉, 魏国光, 张阿磊, 等. 一种利用N-乙酰氨基葡萄糖制备 3-氨基-5-(α-氨基乙基)四氢呋喃的方法: CN109824629B[P]. 2022-12-09.

CHEN K Q, WEI G G, ZHANG A L, et al. A method for preparing 3-amino-5-(α-aminoethyl) tetrahydrofuranusing from N-acetylglucosamine: CN109824629B[P]. 2022-12-09.

139

Biocatalytic synthesis of two furan-based amino compounds 2-acetyl-4-aminofuran and 3-acetylamino-5-(α-aminoethyl)-furan from chitin resources
WU C Q, ZHANG X, LIU W, et al. Biocatalytic synthesis of two furan-based amino compounds 2-acetyl-4-aminofuran and 3-acetylamino-5-(α-aminoethyl)-furan from chitin resources[J]. ACS Sustainable Chemistry & Engineering, 2024, 12(30): 11145-11154.

第 1 页 / 共 1 页

2024年第5卷第6期

PDF下载

226

78

引用本文

BibTeX

文章信息
doi: 10.12211/2096-8280.2024-041

接收时间：2024-05-13

首发时间：2025-07-07

出版时间：2024-12-31

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2024-05-13

修回日期：2024-08-04

基金

国家重点研发计划(2021YFA0911400)

国家自然科学基金(22278220)

国家自然科学基金(U21B2097)

江苏省农业自主创新基金(CX(22)3070)

作者信息

南京工业大学生物与制药工程学院，材料化学工程国家重点实验室，江苏南京 211816

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/hcsw/CN/10.12211/2096-8280.2024-041

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科
Family 属数
Number of
genus 种数
Number of
species 占总种数比例
Percentage of
total species (%) 属
Genus 种数
Number of
species 占总种数比例
Percentage of total
species (%)

鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78

小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39

多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39

红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87

小菇属 Mycena 11 5.26

光柄菇属 Pluteus 5 2.39

红菇属 Russula 17 8.13

栓菌属 Trametes 5 2.39

关闭全屏

BibTeX

EndNote

RefWorks

TxT

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

生物类型	来源分类	几丁质含量
节肢动物	甲壳纲：虾、蟹等	20%～85%
昆虫纲：蝗虫/蝴蝶/蚊/蛾/蝇/蚕等蛹壳中	20%～60%
多足/蛛形纲：马陆、蜈蚣、蜘蛛、蝎子、螨虫等	4%～22%
软体动物	双神经/腹足/掘足/瓣鳃/头足纲：鲍鱼、蜗牛、角贝、牡蛎、乌贼等	3%～26%
环节动物	原环虫/毛足纲：角涡虫、沙蚕、蚯蚓等	20%～38%
原生动物	鞭毛虫/肉足/孢子虫/纤毛虫纲：锥体虫、变形虫、疟原虫、草履虫等	极少
腔肠动物	水螅虫/钵水母/珊瑚虫纲：水螅、筒螅、海月水母、海蜇、霞水母等	3%～30%
海藻	主要是绿藻	少量
真菌	囊菌、担子菌、藻菌等	微量至45%
动物关节	蹄、足的坚硬部分、动物肌肉、骨结合处等	少量

生物类型

来源分类

几丁质含量

节肢动物

甲壳纲：虾、蟹等

20%～85%

昆虫纲：蝗虫/蝴蝶/蚊/蛾/蝇/蚕等蛹壳中

20%～60%

多足/蛛形纲：马陆、蜈蚣、蜘蛛、蝎子、螨虫等

4%～22%

软体动物

双神经/腹足/掘足/瓣鳃/头足纲：鲍鱼、蜗牛、角贝、牡蛎、乌贼等

3%～26%

环节动物

原环虫/毛足纲：角涡虫、沙蚕、蚯蚓等

20%～38%

原生动物

鞭毛虫/肉足/孢子虫/纤毛虫纲：锥体虫、变形虫、疟原虫、草履虫等

极少

腔肠动物

水螅虫/钵水母/珊瑚虫纲：水螅、筒螅、海月水母、海蜇、霞水母等

3%～30%

海藻

主要是绿藻

少量

真菌

囊菌、担子菌、藻菌等

微量至45%

动物关节

蹄、足的坚硬部分、动物肌肉、骨结合处等

少量

酶	活性	酶活	参考文献
RFChiA	持续性外切	6.9 U/mg	[75]
Chit46	内切	9.5 U/mg	[76]
ActChi	持续性外切	3.7 U/mg	[64]
Chi304	内切、外切	ND	[65]
Chit33	内切	2.7 U/mg	[77]
R-SaChiA4	内切	28 U/mg	[74]
CmChi1	内切、外切、N-乙酰氨基葡萄糖苷酶	1.1 U/mg	[78]

酶

活性

酶活

参考文献

RFChiA

持续性外切

6.9 U/mg

[75]

Chit46

内切

9.5 U/mg

[76]

ActChi

持续性外切

3.7 U/mg

[64]

Chi304

内切、外切

[65]

Chit33

内切

2.7 U/mg

[77]

R-SaChiA4

内切

28 U/mg

[74]

CmChi1

内切、外切、N-乙酰氨基葡萄糖苷酶

1.1 U/mg

[78]

来源	酶	底物	产物	寡糖产率/产量	参考文献
Salinivibrio sp. BAO-1801	野生几丁质酶	胶体几丁质	几丁二糖，少量GlcNAc	71.5%	[97]
T. gamsii R1	野生几丁质酶	胶体几丁质	几丁二糖、三糖	11.62 g/L，1.92 g/L	[98]
S. marcescens	SmChiB	胶体几丁质	几丁二糖	2.04 g/L	[83]
堆肥宏基因组	ActChi	粉粒几丁质	几丁二糖	17%	[64]
T. harzianum	rChit46	胶体几丁质	几丁二糖，微量GlcNAc	94.8%	[76]
P. barengoltzii	PbChi70	胶体几丁质	几丁二糖，微量GlcNAc	21.6 g/L，89.5%	[68]
F. johnsoniae UW101	FjChiB	胶体几丁质	几丁二糖、三糖	—	[63]
S. marcescens	rCHI-2	胶体几丁质	几丁二糖，微量GlcNAc	—	[99]
M. thermophila C1	Chi1	溶胀几丁质	几丁二糖，微量GlcNAc	—	[100]
A. fumigatus df347	AfChi28	胶体几丁质	几丁二糖到几丁四糖	—	[101]
A. media CZW001	AmChi	粉粒几丁质	几丁五糖、六糖	—	[102]
B. aryabhattai	BaChiA	粉粒几丁质	几丁二糖到几丁六糖	—	[103]
Corallococcus sp. EGB	CcCti1	胶体几丁质	几丁二糖到几丁六糖	—	[39]

来源

酶

底物

产物

寡糖产率/产量

参考文献

Salinivibrio sp. BAO-1801

野生几丁质酶

胶体几丁质

几丁二糖，少量GlcNAc

71.5%

[97]

T. gamsii R1

野生几丁质酶

胶体几丁质

几丁二糖、三糖

11.62 g/L，1.92 g/L

[98]

S. marcescens

SmChiB

胶体几丁质

几丁二糖

2.04 g/L

[83]

堆肥宏基因组

ActChi

粉粒几丁质

几丁二糖

17%

[64]

T. harzianum

rChit46

胶体几丁质

几丁二糖，微量GlcNAc

94.8%

[76]

P. barengoltzii

PbChi70

胶体几丁质

几丁二糖，微量GlcNAc

21.6 g/L，89.5%

[68]

F. johnsoniae UW101

FjChiB

胶体几丁质

几丁二糖、三糖

—

[63]

S. marcescens

rCHI-2

胶体几丁质

几丁二糖，微量GlcNAc

—

[99]

M. thermophila C1

Chi1

溶胀几丁质

几丁二糖，微量GlcNAc

—

[100]

A. fumigatus df347

AfChi28

胶体几丁质

几丁二糖到几丁四糖

—

[101]

A. media CZW001

AmChi

粉粒几丁质

几丁五糖、六糖

—

[102]

B. aryabhattai

BaChiA

粉粒几丁质

几丁二糖到几丁六糖

—

[103]

Corallococcus sp. EGB

CcCti1

胶体几丁质

几丁二糖到几丁六糖

—

[39]

酶（来源）	底物	GlcNAc浓度/（g/L）	转化率	参考文献
C. meiyuanensis SYBC-H1发酵液	粉粒几丁质	39.3	98%	[111]
微生物发酵处理几丁质	19.2	96%	[112]
超声处理几丁质	2.65	100%	[113]
高压均质小龙虾壳	3.9	—	[114]
有机溶剂预处理几丁质	4.6～7.6	96%	[115]
碱冻融处理几丁质	75	98%	[116]
A. caviae CH129发酵液	胶体几丁质	—	93%	[117]
A. terreus 发酵液	膨胀几丁质	46	92%	[67]
S. proteamaculans NJ303发酵液	高压均质小龙虾壳	3.9	78%	[114]
T. harzianum发酵液	冻干几丁质粉	14	80%	[118]
S. albolongus 发酵液	胶体几丁质	4.4	89%	[119]
ScChiC, ScHEX	粉粒几丁质	9.4	94%	[120]
SaChiA4, SvNag2557	胶体几丁质	8.0	80%	[121]
ChiA, BsNagZ	胶体几丁质	—	88%	[122]
BpChiA, BlNagZ	胶体几丁质	—	64%	[123]
CmChi1	胶体几丁质	9.8	98%	[78]
ChiG	胶体几丁质	—	—	[124]
AMCase	胶体几丁质	1.2	87%	[125]
PbChi70突变体, NAGase	胶体几丁质	—	97%	[126]
PbChi74, NAGase	胶体几丁质	27.8	93%	[127]

酶（来源）

底物

GlcNAc浓度/（g/L）

转化率

参考文献

C. meiyuanensis

SYBC-H1发酵液

粉粒几丁质

39.3

98%

[111]

微生物发酵处理几丁质